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Silver nitrate-positive opines in crown gall tumors
Plant Science Letters, 32 (1983) 193--203 Elsevier Scientific Publishers Ireland Ltd.
193
SILVER NITRATE-POSITIVE OPINES IN CROWN GALL TUMORS
G.A. DAHL*, P. GUYON, A. PETIT and J. TEMP]~ Institut de Microbiologie, Universitd de Paris-Sud, 91405 Orsay (France) (Received January 1st, 1983) (Revision received April 6th, 1983) (Accepted May 17th, 1983)
SUMMARY
Agrobacterium tumefaciens strains harboring octopine- or agropine-type Ti-plasmids induce crown gall tumors on Kalancho~ tubiflora, sunflower or carrot, that contain agropine, mannopine, mannopinic acid, and agropinic acid. Since oxidation of these compounds on electrophoretograms by an alkaline silver nitrate reagent yields darkly stained spots due to reduction of the silver ions, they are called 'silver nitrate-positive'. These silver nitratepositive compounds are not present in normal plant tissues nor in tumors induced by A. tumefaciens strains that contain a nopaline-type Ti-plasmid. All four compounds are opines since octopine- and agropine-type Ti-plasmids, but not nopaline-type Ti-plasmids, confer on the bacterial host the ability to catabolize them for growth. Silver nitrate-positive opines were always present in primary octopine- or agropine-type crown gall tumors. However, cultured crown gall tumors frequently did not contain detectable silver nitrate-positive opines even though other types of opines, such as octopine, were usually present. Key words: O p i n e s - Crown gall tumors--Agrobacterium tumefaciens
INTRODUCTION
Most A. tumefaciens strains, Ti-plasmids, and crown gall tumors can be classified into one of three opine types (Table I) according to the opines that are in the tumors and which are degraded by the Ti-plasmid catabolic functions [ 1--6 ]. Agropine is an opine in high abundance in octopine- and agropine-type *Present address: Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, U.S.A. 0304-4211/83/$03.00 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
194 TABLE I O P I N E S A S S O C I A T E D W I T H D I F F E R E N T T Y P E S O F A. T U M E F A C I E N S Ti-PLASMIDS, A N D C R O W N G A L L T U M O R S
STRAINS,
Data from Refs. 1--6 and this work.
Opine type
Opines present in tumors and degraded by Agrobacteria sp.
Octopine
Lysopine, octopine, octopinic acid, histopine, agropine, mannopine, mannopinic acid, agropinic acid
Nopaline
Nopaline, nopalinic acid, agrocinopine :~
Agropine
Agropine, mannopine, mannopinic acid, agropinic acid, agrocinopine C
crown gall tumors [5,7,8]. This c o m p o u n d , which reacts with alkaline silver nitrate reagent ('silver nitrate-positive'), was also present in hairy roots incited b y some Agrobacteriurn rhizogenes strains [9]. In subsequent studies [10], two types o f A . rhizogenes strains were found: hairy roots incited by 'agropine-type' strains contain agropine, mannopine, mannopinic acid, and agropinic acid as specific silver nitrate-positive substances, while those incited by 'mannopine-type' strains contain mannopine, mannopinic acid, and agropinic acid, b u t n o t agropine. The present paper reports the results o f investigations on the contents of silver nitrate-positive opines in crown gall tumors incited by many strains o f A. tumefaciens. These studies were undertaken to further define the crown gall system with respect to silver nitrate-positive opines and to look for differences in opine t y p e s similar to those found in the hairy r o o t system. The abilities of the various silver nitrate-positive substances to serve as specific growth substrates for A. tumefaciens strains are also reported and are correlated with opine consumption studies. Many recent reviews related to the crown gall system can be found in Ref. 11. MATERIALS AND METHODS
Bacterial strains The bacterial strains used in this s t u d y are listed in Table II. Transconjugant strains were obtained as follows: Ti-plasmid-containing donor and Ti-plasmid-free receptor strains were grown overnight in liquid selective or mannitol medium to an optical density (680 nm) of 0.6--1. Then drops of the d o n o r culture were spotted onto a freshly-plated, b u t dry, lawn of the recipient on medium that contained an opine (2 g o f octopine, nopaline,
195 TABLE II
A. TUMEFACIENS STRAINS USED IN THIS STUDY Sources: 1, J.L. Firmin (John Innes Institute, Norwich, U.K.); 2, P. Manigault (Institut Pasteur, Paris); 3, J. Schell (Max Planck Institute, Cologne); 4, A. Faivre Amyot (CNRA, Versailles); 5, R. Beardsley (Manhattan College, New York); 6, This laboratory; 7, E.W. Nester (Univ. of Washington, Seattle); 8, M. Rafaila; 9, American Type Culture Collection (Rockville, MD); 10, A. Kerr (Waite Institute, Adelaide); 11, G. Melchers (Max Planck Institute, Turbingen); 12, B. Digat (INRA, Angers, France). Strain
Source
Octopine-type A6 A66 ACH5 B6S3 B6-806 B91
BP C58C1:pTiB6S3 C58C1:pTiR10 CGIC RIO
R32 $56 $341 SP W1 15955 89(10)
1 2 3 3 2 4 5
6 6 7 8
8 4 2 5 5 9 10
Nopaline-type C58 C58C1 : pTiT37 H100 T37 113.25 II-BV7
3 6 5 2 9 2
Agropine-type Bo542 C58C1:pTiBo542 Dahlia
11 6 12
Ti-plasmid-cured C58C1
3
o r m a n n o p i n i c acid/l) t h a t c o u l d b e c a t a b o l i z e d b y t h e d o n o r strain a n d t h e a n t i b i o t i c s t o w h i c h t h e A. tumefaciens strain C 5 8 C 1 r e c i p i e n t was r e s i s t a n t . T r a n s c o n j u g a n t c o l o n i e s t h a t a p p e a r e d a f t e r 4 - - 5 d a y s w e r e p u r i f i e d b y rep e a t e d s t r e a k i n g a n d g r o w t h o n selective m e d i u m a n d w e r e i d e n t i f i e d b y their phage type [12].
196
Bacterial culture media The bacterial culture media were as described [13] except that mannitol (2 g/l) was used instead o f glucose as the non-selective carbon source. In order to test a substance as a selective growth substrate, a neutralized solution o f the substance was filter-sterilized and added to the medium in place o f mannitol at a final concentration of 2.g/1. Mannitol was replaced by a t u m o r extract for studies on the consumption of the silver nitrate-positive substances. For nutritional studies with solidified medium, Difco Nobel Agar (16 g/l) was used because it gave minimal background growth.
Crown gall tumor lines The lines of crown gall tumors t h a t were used in this study are given in Table III.
Plant tissue culture media Primary tumors t h a t were p u t into culture and lines of crown gall tumors were grown on Linsmaier and Skoog medium [14] or on Monnier medium [15] t h a t was supplemented with the vitamin mix o f Morel and Wetmore [16] and sucrose (30 g/l). Carbenicillin (0.5--1 mg/ml) was sometimes added to the medium for the first two or three transfers. The tissues were generally grown in diffuse light (16 h / d a y ) at room temperature and were routinely transferred to fresh m e d i u m at 5--8-week intervals.
Induction o f tumors Crown gall tumors were induced on Helianthus annuus (sunflower) cv. INRA 6501 hypocotyls and on K. tubiflora plants as previously described [13] and were taken for analysis after 4--7 weeks o f growth. Tumors were induced on discs of carrots (Daucus carota) from the market: The carrots were washed with water, sterilized by immersion in 6% sodium hypochlorite for 15--30 rain, rinsed with sterile water, and aseptically cut into 3-ramthick discs that were planted on agar-solidified tap water. Bacteria from a fresh culture of an A. tumefaciens strain were suspended in sterile distilled water to a b o u t 109 ceUs/ml and about 0.05 ml of this suspension was spread on the surface o f each carrot disc. Tumorous overgrowths were generally seen after about 2 weeks and were taken for analysis after about 8 weeks.
Preparation o f tumor samples for opine analysis A b o u t 0.5--1 g of tissue from a newly-induced (primary) t u m o r or from a t u m o r line was macerated with a glass rod in a 1.5-ml Eppendorf tube. After centrifugation for 2 min, the supernatant was used for analysis w i t h o u t further treatment. Occasionally, the tissue was boiled for 7--10 min in 0.12 N HC1 (1 ml/g tissue), macerated, centrifuged, and the supernatant, kept at 40°C, was evaporated to dryness under a stream of air; the residue
197' TABLE III PLANT TISSUE LINES USED IN THIS STUDY Sources: 1, H. annuus cv. Russian Mammoth; J.D. Kemp, Univ. of Wisconsin, Madison; 2, H. annuus cv. INRA 6501, this work; 3, Nicotiana tabacum cv. Xanthi, M.P. Gordon, Univ..of Washington, Seattle; 4, N. tabacum cv. White Burley; J.L. Firmin, John Innes Institute, Norwich, U.K.; 5, N. tabacum cv. Wisconsin 3 8 ; J . Tourneur, CNRA, Versailles; 6, N. tabacum cv. Havana; A. Braun, Rockefeller Univ., New York; 7, N. tabacum cv. Samsun; G. Melchers, Max Planck Institut, Tubingen. Line
Plant material and source
Inciting A. tumefaciens strain
Octopine-type PSCG-B6 PSCG-15955 ts A66 ts R32 ts 15955 (10 independent fines) 15955/1 15955/01 A66 W-A6 W-B634
1 1 2 2 2 3 3 4 5 5
B6 15955 A66 R32 15955 15955 15955 A66 A6 B634
Nopalme-type PSCG-C58 ts 11325 BT37 W-C58 W-T37
1 2 6 5 5
C58 113.25 T37 C58 T37
Agropine-type E228 Bo 542
7 7
Bo542 Bo542
Other HSSS SSS
1 1
None (habituated tissue) None (normal tissue)
was resuspended in 0.2 ml of water/g tissue and clarified by centrifugation before analysis.
Silver nitrate-positive opines Details on the structural identifications and syntheses of the silver nitratepositive opines are presented elsewhere [ 10,17--19 ].
Electrophoresis and analysis of samples. Usually, 2--4 #1 of a t u m o r sample, corresponding to 5--10 mg of tissue, was spotted on Whatman 3 MM paper for analysis, but 20--30 ~1 of sample was used to verify a negative result. For studies on the consumption b y
198
A. tumefaciens strains o f silver nitrate-positive substances in t u m o r extracts, these were added to the culture medium in place of mannitol at a final concentration of 0.5--2 mg/ml, as judged by comparative analyses with known amounts o f synthetic materials; then, 4--6 pl o f a culture was spotted at various times after inoculation with a bacterial strain. The Whatman 3MM paper with the samples was carefully wetted with the electrophoresis buffer and submitted to electrophoresls (Gflson High Voltage Electrophoresis Apparatus, Model D) at 50--75 V/cm for 12--20 min. Two different buffers were used: pH approx. 1.9, formic acid/acetic acid/water (3 : 6 : 91, v/v/v) and pH 9.8, 5 g a m m o n i u m carbonate (Prolabo, mixture of a m m o n i u m bicarbonate and a m m o n i u m carbamate)/1 adjusted to pH 9.8 with concentrated a m m o n i u m hydroxide. The electrophoretograms were then dried in a current of hot air; those from the alkaline buffer were additionally dried for 45--60 min in an oven at l l 0 ° C . As described [20], the dry electrophoretograms were dipped in silver nitrate solution (4 g of silver nitrate in 20 ml of water diluted to 1 1 with acetone), dried in a current of cool air, and developed in ethanolic NaOH (2% in 90% ethanol). The papers were allowed to stand just until dry, whereupon t h e y were dipped in fixer (Kodak X-ray fixer AL 4) and immediately washed for several hours in running water. The darkly staining substances were designated 'silver nitrate-positive substances'. Agropine, mannopine , mannopinic acid, and agropinic acid were identified by their electrophoretic mobilities [10] and were quantified 5ased on their silver nitrate staining densities relative to the staining densities of known concentrations of authentic standards. Electrophoretograms of some samples were stained for the presence of octopine or nopaline with the phenanthrene quinone reagent [21]. The amounts of nopaline in nopaline-type tumors were usually sufficiently high that this opine could also be detected as a negatively-stained (i.e., lighter than background) spot on silver nitrate-stained electrophoretograms. RESULTS
Silver nitrate-positive substances in primary tumors Every one of the octopine-type or agropine-type A. tumefaciens strains listed in Table II induced tumors on sunflower hypocotyls, on carrot discs or on K. tubiflora plants that contained all o f the following silver nitratepositive substances: agropine; mannopine, N 2-(1-D-mannityl-)-L-glutamine; mannopinic acid, AP -( 1-D-mannityl- ) -L-glutamic acid; agropinic acid, N-( 1 -Dmannityl-)-5-carboxy-pyrrolidine-2-one. Conjugative transfer of the pTiB6S3 or the pTiR10 octopine plasmids or of the pTiBo542 agropine plasmid to the Ti-plasmid-cured. A. tumefaciens strain C58C1 also conferred on this recipient the ability to induce tumors that contained these silver nitratepositive substances. The concentrations of silver nitrate-positive substances in primary tumors were variable whether the tumors were induced on a given plant species by the same A. tumefaciens strain or by a different strain, but
199
generally fell within the range of 0.05--1% o f the fresh wt. of the tissue analyzed. Under the conditions used, none of the primary tumors induced b y nopaline-type A. tumefaciens strains (Table II) contained any detectable a m o u n t of specific silver nitrate-positive substances, although all of them contained nopaline. Normal .plant materials were also always negative for these substances.
Silver nitrate-positive substances in tumor lines Although each o f the approx. 50 primary tumors induced on K. tubiflora plants, sunflower h y p o c o t y l s and carrot discs b y octopine- or agropine-type A. tumefaciens strains contained all 4 silver nitrate-positive substances, many bacteria-free lines established from tumors incited b y some o f the same bacterial strains (Table III) did n o t contain detectable levels of any of these substances {Fig. 1). Some t u m o r lines continued to synthesize very high levels (up to several percent of the fresh wt.) of the silver nitrate-positive substances, however, and other lines synthesized lesser amounts. Ten lines established from different tumors induced on sunflower b y the same octopine-type A. tumefaciens strain 15955 contained levels o f the silver nitrate-positive substances that varied from high to undetectable. Most octopine t u m o r lines that did n o t contain detectable specific silver nitratepositive substances nevertheless did contain octopine. Growth o f A. tumefaciens strains on silver nitrate-positive substances With the exception o f strain R32, all of the A. tumefaciens strains listed in Table II that contained octopine- or agropine-type Ti-plasmids were able to grow on m e d i u m that contained any o f the 4 silver nitrate-positive substances as the sole carbon source. Growth was good on agropine-, mannopine- or mannopinic acid-containing medium within 2--4 days, b u t good growth on agropinic acid~containing medium was observed only after a b o u t 8 days. Octopine-type strain R32, which did n o t grow on any of these media, also did n o t grow on medium that contained mannitol as the carbon source; this strain may be an auxotroph for an essential nutrient since it grew well on medium that contained yeast extract (2 g/l). Nopaline-type A. tumefaciens strains C58, T37, H 100, II-BV7 and 11325, and A. tumefaciens strain C58C1, which does n o t contain a Ti-plasmid, did n o t grow on any of the media that contained a silver nitrate-positive substance as the sole carbon source. Catabolism o f silver nitrate-positive substances by A. tumefaciens strains The catabolism o f silver nitrate-positive substances b y A. tumefaciens strains correlated exactly with their abilities to grow on these substances. Thus, agropine, mannopine, mannopinic acid, and agropinic acid were catabolized b y A. tumefaciens strains that contained octopine- or agropinet y p e Ti-plasmids, b u t n o t b y strains that were cured o f their Ti-plasmid or that contained a nopaline-type Ti-plasmid (for example, see Fig. 2). A.
200
AGROlC~NE
8
A
B
C
S
D
E
F
MANNOPI MANNOPI
NE ÷ NIC ACID
NEUTRAL AGROPINI
COMPDS C ACID
*
S
" MANNOPINIC 9 AGROPINIC
ACID ACID
q MANNOPINE
q NEUTRAL AGROPINE
S
A
B
C
D
E
F
COMPDS
S
Fig. 1. Silver nitrate-positive substances in sunflower crown gall tumor lines, identified after electrophoresis toward the cathode at pH 1.9 (a) or toward the anode at pH 9.8 (b): (A) ts 11325, (B) ts R32, (C) ts A66, (D) ts 15955-10, (E) PSCG-15955, (F) PSCG-B6, (S) chemical standards. The 'ts' and 'PSCG' lines were maintained in culture for one year or several years, respectively. In separate experiments, the non-specific substances on the electrophoretograrn after alkaline electrophoresis were consumed by Ti-plasmidcured A. tumefaciens strain C58C1, indicating that they were not opines.
tumefaciens strain R32, which did n o t grow o n m e d i u m that contained a silver nitrate-positive substance as the sole carbon source, nevertheless did grow and catabolize these substances in m e d i u m that a d d i t i o n a l l y c o n t a i n e d y e a s t e x t r a c t (200 mg/1).
201
AGROPINE
MANNOPINE + MANNOPINIC ACID
NEUTRAL C O ~%IP'DS + AGROPINIC ACID
>
~
>
~
o
~
~
~
o
~
~
~
-
r~J
Fig. 2. Specific catabolism of silver nitrate-positive substances b y A. tumefaciens strains. The indicated strains were each inoculated into medium that contained an extract of the ts 15955-10 tumor line instead of mannitol. After 48 h, the bacteria were pelleted and samples of the medium were spotted for electrophoresis at pH 1.9.
DISCUSSION
The crown gall tumor-specific silvernitrate-positive substances appear to be opines for octopine- and agropine-type A. tumefaciens strains. Thus, agropine, mannopine, mannopinic acid, and agropinic acid can be catabolized and used as the sole carbon source for growth by A. tumefaciens strains that incite tumors that contain these substances. Genes responsible for the catabolism of these silvernitrate-positive opines by the bacteria and genes responsible for their synthesis in crown gall tumors are Ti-plasrnidborne since these traits can be transferred to a Ti-plasrnid-cured strain of A. tumefaciens by conjugation. T h e simplest hypothesis is that a T - D N A - e n c o d e d e n z y m e in the crown gall tumors catalyzes the reduction of the Schiff base formed between L-glutamine and D-mannose to yield mannopine and that a different e n z y m e or a different site on the same e n z y m e catalyzes the cyclization of manno-
202
pine to yield agropine. However, activity of a putative mannopine dehydrogenase could not be detected in extracts of mannopine-containing octopine tumors that had lysopine dehydrogenase activity (our unpublished work; J.D. Kemp, pers. comm.). Agropinic acid, and possibly mannopinic acid, present a new situation with respect to the opine concept. Strictly speaking, agropinic acid could be excluded from this new silver nitrate-positive family of opines: it can be formed by chemical rearrangement of either agropine or mannopine [10, 18,19] and therefore, its synthesis is probably not directly coded for by genetic information from the pathogen. Bacterial catabolism of agropinic acid is Ti-plasmid-encoded, however, since A. tumefaciens strains that contain octopine or agropine Ti-plasmids, but not strains that contain a nopaline Ti-plasmid, are able to catabolize agropinic acid as the sole source of carbon for growth. Thus, in view of the facts that genes responsible for agropinic acid catabolism are Ti-plasmid-borne and that agropinic acid is derived from other substances (agropine or mannopine) whose biosyntheses are Ti-plasmid-encoded (directly or indirectly), we suggest that this substance should be considered an opine. It is not yet known whether mannopinic acid synthesis is coded for by T-DNA in the tumors or results from spontaneous chemical or enzymatic hydrolysis of mannopine. In either case, based on the same reasoning as for agropinic acid, it should also be considered as an opine for octopine- and agropine-type A. tumefaciens strains. All of the analyzed primary tumors incited by octopine- or agropine-type A. tumefaciens strains contained the four silver nitrate=positive compounds. On the other hand, some tissue culture lines of tumors incited by the same A. tumefaciens strains did not contain these opines even though the octopinetype lines did contain octopine; similar results with tumor lines have been observed in other laboratories (J.D. Kemp and J. schell, pets. comm.). The observed variability in the levels and presence of the silver nitrate-positive opines in the tumors probably reflects variability in the T-DNA in the plant tumor cells. Thomashow et al. [22] observed that the right side of the T-DNA (TR-DNA) in octopine tumors was variable, sometimes being absent and sometimes present in many copies. De Beuckeleer et al. [23] reported that neither this TR-DNA nor agropine could be detected in any of the five octopine tumor lines that they studied. Finally, the presence of certain deletions in the TR-DNA region of the pTiACH5 plasmid yielded A. tumefaciens strains which incited tumors that did not contain any silver nitrate-positive opines (Y. Dessaux, pers. comm.). ACKNOWLEDGEMENTS
This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Recherche Agronomique and the Ddldgation Gdndrale ~i la Recherche Scientifique et Technique. G.A.D. was supported by a postdoctoral fellowship No. 1F32-CA06390 awarded by the National Cancer Institute, DHHS, U.S.A.
203
REFERENCES 1 A. Petit, S. Delhaye, J. Temp~ and G. Morel, Physiol. Veg., 8 (1970) 205. 2 G.H. Bomhoff, P.M. Klapwijk, H.C. Kester, R.A. Schilperoort, J.P. Hernalsteens and J. ScheU, Mol. Gen. Gen~t., 145 (1976) 177. 3 A. Montoya, M.D. Chilton, M.P. Gordon, D. Sciaky and E.W. Nester, J. Bacteriol., 129 (1977) 101. 4 J.D. Kemp, Plant Physiol., 62 (1978) 26. 5 P. Guyon, M.D. Chilton, A. Petit and J. Temp~, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 2693. 6 J.G. Ellis and P.J. Murphy, Mol. Gen. Genet., 181 (1981) 36. 7 J.L. Firmin and G.R. Fenwick, Nature (London), 276 (1978) 842. 8 I.M. Scott, J.L. Firmin, D.N. Butcher, L.M. Searle, A.K. Sogeke, J. Eagles, J.F. March, R. Self and G.R. Fenwick, Mol. Gen. Genet.~ 176 (1979) 57. 9 D.A. Tepfer and J. Temp~, C.R. Acad. Sci. Set. III (Paris), 292 (1981) 153. 10 A. Petit, C. David, G.A. Dahl, J.G. Ellis, P. Guyon, F. Casse-Delbart and J. Tempd, Mol. Gen. Genet., 190 (1983) 204. 11 G. Kahl and J.S. Schell (Eds.), Molecular Biology of Plant Tumors, Academic Press, New York 1982. 12 G. Vervliet, M. Holsters, H. Teuchy, M. Van Montagu and J. Schell, J. Gen. Virol., 26 (1974) 33. 13 A. Petit and J. Temp~ Mol. Gen. Genet., 167 (1978) 147. 14 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 15 M. Monnier, Moench. Rev. Cytol. Biol. V~g., 39 (1976) 1. 16 G. More'] and R.H. Wetmore, Am. J. Bot., 38 (1951) 141. 17 J. Temp~, P. Guyon, A. Petit, J.G. Ellis, M.E. Tate and A. Kerr, C.R. Acad. Sci. Ser. D (Paris), 290 (1980) 1173. 18 D.T. Coxon, A.M.C. Davies G.R. Fenwick, R. Self, J.L. Fkmin, D. Lipkin and N.F. Janes. Tetrahedron Lett., 21 (1980) 495. 19 M.E. Tate, J.G. Ellis A. Kerr, J. Temp~, K.E. Murray and K.J. Shaw, Carbohydr. Res., 104 (1982) 105. 20 W.E. Trevelyan, D.P. Procter and J.P. Harrison, Nature (London), 166 (1950) 444. 21 S. Yamada and H.A. Itano, Biochim. Biophys. Acta, 133 (1966) 538. 22 M.F. Thomashow, R. Nutter A.L. Montoya, M.P. Gordon and E.W. Nester, Cell, 19 (1980) 729. 23 M. De Beuckeleer, M. Lemmers G. De Vos, L. Willmitzer, M. Van Montagu and J. Schell, Mol. Gen. Genet., 183 (1981) 283.
193
SILVER NITRATE-POSITIVE OPINES IN CROWN GALL TUMORS
G.A. DAHL*, P. GUYON, A. PETIT and J. TEMP]~ Institut de Microbiologie, Universitd de Paris-Sud, 91405 Orsay (France) (Received January 1st, 1983) (Revision received April 6th, 1983) (Accepted May 17th, 1983)
SUMMARY
Agrobacterium tumefaciens strains harboring octopine- or agropine-type Ti-plasmids induce crown gall tumors on Kalancho~ tubiflora, sunflower or carrot, that contain agropine, mannopine, mannopinic acid, and agropinic acid. Since oxidation of these compounds on electrophoretograms by an alkaline silver nitrate reagent yields darkly stained spots due to reduction of the silver ions, they are called 'silver nitrate-positive'. These silver nitratepositive compounds are not present in normal plant tissues nor in tumors induced by A. tumefaciens strains that contain a nopaline-type Ti-plasmid. All four compounds are opines since octopine- and agropine-type Ti-plasmids, but not nopaline-type Ti-plasmids, confer on the bacterial host the ability to catabolize them for growth. Silver nitrate-positive opines were always present in primary octopine- or agropine-type crown gall tumors. However, cultured crown gall tumors frequently did not contain detectable silver nitrate-positive opines even though other types of opines, such as octopine, were usually present. Key words: O p i n e s - Crown gall tumors--Agrobacterium tumefaciens
INTRODUCTION
Most A. tumefaciens strains, Ti-plasmids, and crown gall tumors can be classified into one of three opine types (Table I) according to the opines that are in the tumors and which are degraded by the Ti-plasmid catabolic functions [ 1--6 ]. Agropine is an opine in high abundance in octopine- and agropine-type *Present address: Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, U.S.A. 0304-4211/83/$03.00 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
194 TABLE I O P I N E S A S S O C I A T E D W I T H D I F F E R E N T T Y P E S O F A. T U M E F A C I E N S Ti-PLASMIDS, A N D C R O W N G A L L T U M O R S
STRAINS,
Data from Refs. 1--6 and this work.
Opine type
Opines present in tumors and degraded by Agrobacteria sp.
Octopine
Lysopine, octopine, octopinic acid, histopine, agropine, mannopine, mannopinic acid, agropinic acid
Nopaline
Nopaline, nopalinic acid, agrocinopine :~
Agropine
Agropine, mannopine, mannopinic acid, agropinic acid, agrocinopine C
crown gall tumors [5,7,8]. This c o m p o u n d , which reacts with alkaline silver nitrate reagent ('silver nitrate-positive'), was also present in hairy roots incited b y some Agrobacteriurn rhizogenes strains [9]. In subsequent studies [10], two types o f A . rhizogenes strains were found: hairy roots incited by 'agropine-type' strains contain agropine, mannopine, mannopinic acid, and agropinic acid as specific silver nitrate-positive substances, while those incited by 'mannopine-type' strains contain mannopine, mannopinic acid, and agropinic acid, b u t n o t agropine. The present paper reports the results o f investigations on the contents of silver nitrate-positive opines in crown gall tumors incited by many strains o f A. tumefaciens. These studies were undertaken to further define the crown gall system with respect to silver nitrate-positive opines and to look for differences in opine t y p e s similar to those found in the hairy r o o t system. The abilities of the various silver nitrate-positive substances to serve as specific growth substrates for A. tumefaciens strains are also reported and are correlated with opine consumption studies. Many recent reviews related to the crown gall system can be found in Ref. 11. MATERIALS AND METHODS
Bacterial strains The bacterial strains used in this s t u d y are listed in Table II. Transconjugant strains were obtained as follows: Ti-plasmid-containing donor and Ti-plasmid-free receptor strains were grown overnight in liquid selective or mannitol medium to an optical density (680 nm) of 0.6--1. Then drops of the d o n o r culture were spotted onto a freshly-plated, b u t dry, lawn of the recipient on medium that contained an opine (2 g o f octopine, nopaline,
195 TABLE II
A. TUMEFACIENS STRAINS USED IN THIS STUDY Sources: 1, J.L. Firmin (John Innes Institute, Norwich, U.K.); 2, P. Manigault (Institut Pasteur, Paris); 3, J. Schell (Max Planck Institute, Cologne); 4, A. Faivre Amyot (CNRA, Versailles); 5, R. Beardsley (Manhattan College, New York); 6, This laboratory; 7, E.W. Nester (Univ. of Washington, Seattle); 8, M. Rafaila; 9, American Type Culture Collection (Rockville, MD); 10, A. Kerr (Waite Institute, Adelaide); 11, G. Melchers (Max Planck Institute, Turbingen); 12, B. Digat (INRA, Angers, France). Strain
Source
Octopine-type A6 A66 ACH5 B6S3 B6-806 B91
BP C58C1:pTiB6S3 C58C1:pTiR10 CGIC RIO
R32 $56 $341 SP W1 15955 89(10)
1 2 3 3 2 4 5
6 6 7 8
8 4 2 5 5 9 10
Nopaline-type C58 C58C1 : pTiT37 H100 T37 113.25 II-BV7
3 6 5 2 9 2
Agropine-type Bo542 C58C1:pTiBo542 Dahlia
11 6 12
Ti-plasmid-cured C58C1
3
o r m a n n o p i n i c acid/l) t h a t c o u l d b e c a t a b o l i z e d b y t h e d o n o r strain a n d t h e a n t i b i o t i c s t o w h i c h t h e A. tumefaciens strain C 5 8 C 1 r e c i p i e n t was r e s i s t a n t . T r a n s c o n j u g a n t c o l o n i e s t h a t a p p e a r e d a f t e r 4 - - 5 d a y s w e r e p u r i f i e d b y rep e a t e d s t r e a k i n g a n d g r o w t h o n selective m e d i u m a n d w e r e i d e n t i f i e d b y their phage type [12].
196
Bacterial culture media The bacterial culture media were as described [13] except that mannitol (2 g/l) was used instead o f glucose as the non-selective carbon source. In order to test a substance as a selective growth substrate, a neutralized solution o f the substance was filter-sterilized and added to the medium in place o f mannitol at a final concentration of 2.g/1. Mannitol was replaced by a t u m o r extract for studies on the consumption of the silver nitrate-positive substances. For nutritional studies with solidified medium, Difco Nobel Agar (16 g/l) was used because it gave minimal background growth.
Crown gall tumor lines The lines of crown gall tumors t h a t were used in this study are given in Table III.
Plant tissue culture media Primary tumors t h a t were p u t into culture and lines of crown gall tumors were grown on Linsmaier and Skoog medium [14] or on Monnier medium [15] t h a t was supplemented with the vitamin mix o f Morel and Wetmore [16] and sucrose (30 g/l). Carbenicillin (0.5--1 mg/ml) was sometimes added to the medium for the first two or three transfers. The tissues were generally grown in diffuse light (16 h / d a y ) at room temperature and were routinely transferred to fresh m e d i u m at 5--8-week intervals.
Induction o f tumors Crown gall tumors were induced on Helianthus annuus (sunflower) cv. INRA 6501 hypocotyls and on K. tubiflora plants as previously described [13] and were taken for analysis after 4--7 weeks o f growth. Tumors were induced on discs of carrots (Daucus carota) from the market: The carrots were washed with water, sterilized by immersion in 6% sodium hypochlorite for 15--30 rain, rinsed with sterile water, and aseptically cut into 3-ramthick discs that were planted on agar-solidified tap water. Bacteria from a fresh culture of an A. tumefaciens strain were suspended in sterile distilled water to a b o u t 109 ceUs/ml and about 0.05 ml of this suspension was spread on the surface o f each carrot disc. Tumorous overgrowths were generally seen after about 2 weeks and were taken for analysis after about 8 weeks.
Preparation o f tumor samples for opine analysis A b o u t 0.5--1 g of tissue from a newly-induced (primary) t u m o r or from a t u m o r line was macerated with a glass rod in a 1.5-ml Eppendorf tube. After centrifugation for 2 min, the supernatant was used for analysis w i t h o u t further treatment. Occasionally, the tissue was boiled for 7--10 min in 0.12 N HC1 (1 ml/g tissue), macerated, centrifuged, and the supernatant, kept at 40°C, was evaporated to dryness under a stream of air; the residue
197' TABLE III PLANT TISSUE LINES USED IN THIS STUDY Sources: 1, H. annuus cv. Russian Mammoth; J.D. Kemp, Univ. of Wisconsin, Madison; 2, H. annuus cv. INRA 6501, this work; 3, Nicotiana tabacum cv. Xanthi, M.P. Gordon, Univ..of Washington, Seattle; 4, N. tabacum cv. White Burley; J.L. Firmin, John Innes Institute, Norwich, U.K.; 5, N. tabacum cv. Wisconsin 3 8 ; J . Tourneur, CNRA, Versailles; 6, N. tabacum cv. Havana; A. Braun, Rockefeller Univ., New York; 7, N. tabacum cv. Samsun; G. Melchers, Max Planck Institut, Tubingen. Line
Plant material and source
Inciting A. tumefaciens strain
Octopine-type PSCG-B6 PSCG-15955 ts A66 ts R32 ts 15955 (10 independent fines) 15955/1 15955/01 A66 W-A6 W-B634
1 1 2 2 2 3 3 4 5 5
B6 15955 A66 R32 15955 15955 15955 A66 A6 B634
Nopalme-type PSCG-C58 ts 11325 BT37 W-C58 W-T37
1 2 6 5 5
C58 113.25 T37 C58 T37
Agropine-type E228 Bo 542
7 7
Bo542 Bo542
Other HSSS SSS
1 1
None (habituated tissue) None (normal tissue)
was resuspended in 0.2 ml of water/g tissue and clarified by centrifugation before analysis.
Silver nitrate-positive opines Details on the structural identifications and syntheses of the silver nitratepositive opines are presented elsewhere [ 10,17--19 ].
Electrophoresis and analysis of samples. Usually, 2--4 #1 of a t u m o r sample, corresponding to 5--10 mg of tissue, was spotted on Whatman 3 MM paper for analysis, but 20--30 ~1 of sample was used to verify a negative result. For studies on the consumption b y
198
A. tumefaciens strains o f silver nitrate-positive substances in t u m o r extracts, these were added to the culture medium in place of mannitol at a final concentration of 0.5--2 mg/ml, as judged by comparative analyses with known amounts o f synthetic materials; then, 4--6 pl o f a culture was spotted at various times after inoculation with a bacterial strain. The Whatman 3MM paper with the samples was carefully wetted with the electrophoresis buffer and submitted to electrophoresls (Gflson High Voltage Electrophoresis Apparatus, Model D) at 50--75 V/cm for 12--20 min. Two different buffers were used: pH approx. 1.9, formic acid/acetic acid/water (3 : 6 : 91, v/v/v) and pH 9.8, 5 g a m m o n i u m carbonate (Prolabo, mixture of a m m o n i u m bicarbonate and a m m o n i u m carbamate)/1 adjusted to pH 9.8 with concentrated a m m o n i u m hydroxide. The electrophoretograms were then dried in a current of hot air; those from the alkaline buffer were additionally dried for 45--60 min in an oven at l l 0 ° C . As described [20], the dry electrophoretograms were dipped in silver nitrate solution (4 g of silver nitrate in 20 ml of water diluted to 1 1 with acetone), dried in a current of cool air, and developed in ethanolic NaOH (2% in 90% ethanol). The papers were allowed to stand just until dry, whereupon t h e y were dipped in fixer (Kodak X-ray fixer AL 4) and immediately washed for several hours in running water. The darkly staining substances were designated 'silver nitrate-positive substances'. Agropine, mannopine , mannopinic acid, and agropinic acid were identified by their electrophoretic mobilities [10] and were quantified 5ased on their silver nitrate staining densities relative to the staining densities of known concentrations of authentic standards. Electrophoretograms of some samples were stained for the presence of octopine or nopaline with the phenanthrene quinone reagent [21]. The amounts of nopaline in nopaline-type tumors were usually sufficiently high that this opine could also be detected as a negatively-stained (i.e., lighter than background) spot on silver nitrate-stained electrophoretograms. RESULTS
Silver nitrate-positive substances in primary tumors Every one of the octopine-type or agropine-type A. tumefaciens strains listed in Table II induced tumors on sunflower hypocotyls, on carrot discs or on K. tubiflora plants that contained all o f the following silver nitratepositive substances: agropine; mannopine, N 2-(1-D-mannityl-)-L-glutamine; mannopinic acid, AP -( 1-D-mannityl- ) -L-glutamic acid; agropinic acid, N-( 1 -Dmannityl-)-5-carboxy-pyrrolidine-2-one. Conjugative transfer of the pTiB6S3 or the pTiR10 octopine plasmids or of the pTiBo542 agropine plasmid to the Ti-plasmid-cured. A. tumefaciens strain C58C1 also conferred on this recipient the ability to induce tumors that contained these silver nitratepositive substances. The concentrations of silver nitrate-positive substances in primary tumors were variable whether the tumors were induced on a given plant species by the same A. tumefaciens strain or by a different strain, but
199
generally fell within the range of 0.05--1% o f the fresh wt. of the tissue analyzed. Under the conditions used, none of the primary tumors induced b y nopaline-type A. tumefaciens strains (Table II) contained any detectable a m o u n t of specific silver nitrate-positive substances, although all of them contained nopaline. Normal .plant materials were also always negative for these substances.
Silver nitrate-positive substances in tumor lines Although each o f the approx. 50 primary tumors induced on K. tubiflora plants, sunflower h y p o c o t y l s and carrot discs b y octopine- or agropine-type A. tumefaciens strains contained all 4 silver nitrate-positive substances, many bacteria-free lines established from tumors incited b y some o f the same bacterial strains (Table III) did n o t contain detectable levels of any of these substances {Fig. 1). Some t u m o r lines continued to synthesize very high levels (up to several percent of the fresh wt.) of the silver nitrate-positive substances, however, and other lines synthesized lesser amounts. Ten lines established from different tumors induced on sunflower b y the same octopine-type A. tumefaciens strain 15955 contained levels o f the silver nitrate-positive substances that varied from high to undetectable. Most octopine t u m o r lines that did n o t contain detectable specific silver nitratepositive substances nevertheless did contain octopine. Growth o f A. tumefaciens strains on silver nitrate-positive substances With the exception o f strain R32, all of the A. tumefaciens strains listed in Table II that contained octopine- or agropine-type Ti-plasmids were able to grow on m e d i u m that contained any o f the 4 silver nitrate-positive substances as the sole carbon source. Growth was good on agropine-, mannopine- or mannopinic acid-containing medium within 2--4 days, b u t good growth on agropinic acid~containing medium was observed only after a b o u t 8 days. Octopine-type strain R32, which did n o t grow on any of these media, also did n o t grow on medium that contained mannitol as the carbon source; this strain may be an auxotroph for an essential nutrient since it grew well on medium that contained yeast extract (2 g/l). Nopaline-type A. tumefaciens strains C58, T37, H 100, II-BV7 and 11325, and A. tumefaciens strain C58C1, which does n o t contain a Ti-plasmid, did n o t grow on any of the media that contained a silver nitrate-positive substance as the sole carbon source. Catabolism o f silver nitrate-positive substances by A. tumefaciens strains The catabolism o f silver nitrate-positive substances b y A. tumefaciens strains correlated exactly with their abilities to grow on these substances. Thus, agropine, mannopine, mannopinic acid, and agropinic acid were catabolized b y A. tumefaciens strains that contained octopine- or agropinet y p e Ti-plasmids, b u t n o t b y strains that were cured o f their Ti-plasmid or that contained a nopaline-type Ti-plasmid (for example, see Fig. 2). A.
200
AGROlC~NE
8
A
B
C
S
D
E
F
MANNOPI MANNOPI
NE ÷ NIC ACID
NEUTRAL AGROPINI
COMPDS C ACID
*
S
" MANNOPINIC 9 AGROPINIC
ACID ACID
q MANNOPINE
q NEUTRAL AGROPINE
S
A
B
C
D
E
F
COMPDS
S
Fig. 1. Silver nitrate-positive substances in sunflower crown gall tumor lines, identified after electrophoresis toward the cathode at pH 1.9 (a) or toward the anode at pH 9.8 (b): (A) ts 11325, (B) ts R32, (C) ts A66, (D) ts 15955-10, (E) PSCG-15955, (F) PSCG-B6, (S) chemical standards. The 'ts' and 'PSCG' lines were maintained in culture for one year or several years, respectively. In separate experiments, the non-specific substances on the electrophoretograrn after alkaline electrophoresis were consumed by Ti-plasmidcured A. tumefaciens strain C58C1, indicating that they were not opines.
tumefaciens strain R32, which did n o t grow o n m e d i u m that contained a silver nitrate-positive substance as the sole carbon source, nevertheless did grow and catabolize these substances in m e d i u m that a d d i t i o n a l l y c o n t a i n e d y e a s t e x t r a c t (200 mg/1).
201
AGROPINE
MANNOPINE + MANNOPINIC ACID
NEUTRAL C O ~%IP'DS + AGROPINIC ACID
>
~
>
~
o
~
~
~
o
~
~
~
-
r~J
Fig. 2. Specific catabolism of silver nitrate-positive substances b y A. tumefaciens strains. The indicated strains were each inoculated into medium that contained an extract of the ts 15955-10 tumor line instead of mannitol. After 48 h, the bacteria were pelleted and samples of the medium were spotted for electrophoresis at pH 1.9.
DISCUSSION
The crown gall tumor-specific silvernitrate-positive substances appear to be opines for octopine- and agropine-type A. tumefaciens strains. Thus, agropine, mannopine, mannopinic acid, and agropinic acid can be catabolized and used as the sole carbon source for growth by A. tumefaciens strains that incite tumors that contain these substances. Genes responsible for the catabolism of these silvernitrate-positive opines by the bacteria and genes responsible for their synthesis in crown gall tumors are Ti-plasrnidborne since these traits can be transferred to a Ti-plasrnid-cured strain of A. tumefaciens by conjugation. T h e simplest hypothesis is that a T - D N A - e n c o d e d e n z y m e in the crown gall tumors catalyzes the reduction of the Schiff base formed between L-glutamine and D-mannose to yield mannopine and that a different e n z y m e or a different site on the same e n z y m e catalyzes the cyclization of manno-
202
pine to yield agropine. However, activity of a putative mannopine dehydrogenase could not be detected in extracts of mannopine-containing octopine tumors that had lysopine dehydrogenase activity (our unpublished work; J.D. Kemp, pers. comm.). Agropinic acid, and possibly mannopinic acid, present a new situation with respect to the opine concept. Strictly speaking, agropinic acid could be excluded from this new silver nitrate-positive family of opines: it can be formed by chemical rearrangement of either agropine or mannopine [10, 18,19] and therefore, its synthesis is probably not directly coded for by genetic information from the pathogen. Bacterial catabolism of agropinic acid is Ti-plasmid-encoded, however, since A. tumefaciens strains that contain octopine or agropine Ti-plasmids, but not strains that contain a nopaline Ti-plasmid, are able to catabolize agropinic acid as the sole source of carbon for growth. Thus, in view of the facts that genes responsible for agropinic acid catabolism are Ti-plasmid-borne and that agropinic acid is derived from other substances (agropine or mannopine) whose biosyntheses are Ti-plasmid-encoded (directly or indirectly), we suggest that this substance should be considered an opine. It is not yet known whether mannopinic acid synthesis is coded for by T-DNA in the tumors or results from spontaneous chemical or enzymatic hydrolysis of mannopine. In either case, based on the same reasoning as for agropinic acid, it should also be considered as an opine for octopine- and agropine-type A. tumefaciens strains. All of the analyzed primary tumors incited by octopine- or agropine-type A. tumefaciens strains contained the four silver nitrate=positive compounds. On the other hand, some tissue culture lines of tumors incited by the same A. tumefaciens strains did not contain these opines even though the octopinetype lines did contain octopine; similar results with tumor lines have been observed in other laboratories (J.D. Kemp and J. schell, pets. comm.). The observed variability in the levels and presence of the silver nitrate-positive opines in the tumors probably reflects variability in the T-DNA in the plant tumor cells. Thomashow et al. [22] observed that the right side of the T-DNA (TR-DNA) in octopine tumors was variable, sometimes being absent and sometimes present in many copies. De Beuckeleer et al. [23] reported that neither this TR-DNA nor agropine could be detected in any of the five octopine tumor lines that they studied. Finally, the presence of certain deletions in the TR-DNA region of the pTiACH5 plasmid yielded A. tumefaciens strains which incited tumors that did not contain any silver nitrate-positive opines (Y. Dessaux, pers. comm.). ACKNOWLEDGEMENTS
This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Recherche Agronomique and the Ddldgation Gdndrale ~i la Recherche Scientifique et Technique. G.A.D. was supported by a postdoctoral fellowship No. 1F32-CA06390 awarded by the National Cancer Institute, DHHS, U.S.A.
203
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