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Formation of a growth factor (GABA) by Agrobacterium inoculated leaves and the effect of GABA on tumor initiation and growth
Physiological Plant Patholopv (1976) 9, 331-338
Formation of a growth factor (GABA) by Agrobacferium inoculated leaves and the effect of GABA on tumor initiation and growth K. E. PETERS-~and J. A. LIPPINCOTT Department of Biological Sciences,Northwestern University, Evanston, Illinois 60201, U.S.A. (Acceptedfor publication September1976)
Twenty-seven strains of Agrobacterium, including virulent and avirulent forms, when inoculated on bean leaves induce formation of tumor growth factor II (TGF-II) which has been shown to be y-aminobutyric acid (GABA). Only A. tumefacienrstrain 181 did not; since this strain is highly virulent, formation of TGF-II is not essential for tumor initiation. The level of GABA in bacteria-inoculated leaves is increased about S-fold over wounded controls. Bean leaf tumors show a positive growth response when as little as 0.1 pg of GABA is applied per leaf and this response shows the same high specificity found for animal neuron responses to GABA. Because of the sensitivity of the tumor growth assay and the failure to detect TGF-II activity in extracts of control leaves, it is postulated that a second factor which inhibits tumor response to GABA is present in these extracts but reduced in amount or absent in extracts from bacteriainoculated leaves. Addition of GABA with the bacterial inoculum inhibits tumor initiation.
INTRODUCTION
The isolation of a new crown-gall tumor growth factor (TGF-II) from tumors induced on primary bean leaves by Agrobacterium tumefaciensstrain 13333 was described and evidence presented for its identification as y-aminobutyric acid (GABA) [I?]. This compound may thus be added to a limited and unique group of non-protein amino acids, including octopine and nopaline, which are capable of promoting the growth of bean leaf tumors in uiuo [12, 141. This paper: (a) relates the induction of TGF-II by various strains of Agrobacterium to their virulence and their ability to induce formation of other growth factors and (b) further characterizes the growthpromoting effects of GABA on these tumors. MATERIALS
AND
METHODS
Growth and inoculation of bacteria All strains of Agrobacterium employed in these studies were grown to stationary phase in liquid media as previously described [12]. Primary bean leaves (Phaseolusvulgaris L. var. “Pinto”) were inoculated either in viuo or in vitro, each procedure yielding similar rest&s in terms of the tumor growth activity detected in extracts. The in vivo method of inoculation has been described elsewhere 1121. Briefly, the leaves of t Present address: Department California 91010, U.S.A.
of Medical Genetics, City of Hope National Medical Ccntre, Duarte,
332
K. E. Peters and J. A. Lippincott
young seedlings were spread with O-1 ml of a bacterial suspension (c. 9 x IO* organisms) in the presence of no. 400 grit carborundum by means of a glass rod. The inoculated leaves were harvested 6 to 7 days later for extraction. The in vitro procedures developed by Lippincott & Lippincott (unpublished data) used leaves from 7-day-old seedlings. However, they were removed from the plant, weighed and 10 to 25 g placed in a 15 cm diameter Petri dish. Bacterial inocula (1 ml of stationary phase culture containing c. 9 x lOa bacteria per g of tissue) were added to the dish and the leaves chopped with a razor blade in the presence of the inoculum to yield leaf segments averaging G. 2 x 2 mm. The chopped tissue was then loosely packed in a 250 ml beaker, covered with aluminum foil, and incubated at 27 “C for 18 to 24 h. At this time the tissue was extracted or placed at -20 “C for later extraction. Bacteria for the in vitro inoculations were sedimented by centrifugation at 12 000 g for 20 min, resuspended in 10 mM sodium phosphate buffer, pH 7, and the procedure repeated. The washed bacteria were resuspended in phosphate buffer at their original concentration for inoculation of leaves by chopping. Preparation of extracts Tumor growth factor I (TGF-I), which is believed to be a derivative of histidine [la], was extracted by grinding inoculated leaves in aqueous methanol as described by El Khalifa & Lippincott [6J. Similar procedures were used to extract TGF-II except that 10 mM sodium phosphate buffer, pH 7, was used as the extracting medium [I7]. The extracts were pressed through four layers of cheesecloth, centrifuged at 10 000 g for 15 min at 4 “C. The supernatants containing either TGF-I or TGF-II were then filtered through Whatman no. 1 filter paper and evaporated to dryness in vacuaat 40 “C and redissolved in 1 ml of water per g of initial leaf tissue. To determine the presence of both TGF-I and TGF-II in the same tissue, two extractions were performed in succession using the different solvents: i.e. the residue from the first extraction retained by the cheesecloth plus the precipitate obtained by the initial centrifugation were combined and re-extracted with a second solvent. The order of extraction had no significant qualitative or quantitative effect on the activity detected in the resultant extracts. Tumor growth bioassay The activity of TGF-I, TGF-II and GABA was determined in viva using the bean leaf tumor growth bioassay described by Lippincott & Lippincott [JO]. These data are presented as the mean diameter of the tumors measured for each sample + the standard error of the mean. Where the standard errors are 3- 0.2 S.U. (see Table 1) differences between experimental and control values of O-4 S.U. are significant at the P=O*O5 level and differences of 0.6 S.U. are significant at the P=O*Ol level. Estimation of GABA levels The amount of GABA in leaf extracts was estimated by following NADP reduction in a coupled enzymatic reaction involving deamination of GABA and oxidation of the resultant succinic semi-aldehyde to succinate, This assay is specific for GABA [3]. Prior to assay, the crude TGF-II extracts were partitioned 3 times with ethyl acetate
333
Crown-gall tumors and GABA
and the organic phase containing TGF-II was evaporated to dryness and re-dissolved in water. The TGF-II was then streaked on a preparative silica gel thin-layer chromatography plate (Merck no. 5766) and developed with n-butanol-acetic acid-water (4 : 1 : 1). The silica gel at the R, of GABA was then scraped from the plate and eluted 3 times with water. The eluates were combined, evaporated to dryness and re-dissolved in 1 ml of water. One-tenth ml aliquots, representing c. 2 g initial leaf tissue, were used in the enzymatic assay. The reaction mixture contained the following reagents in a total volume of 3 ml: pyrophosphate buffer, pH 8.1, O-6 mol; 2-mercaptoethanol, 0*01.5 mmol; NADP (sodium salt) pH 7.9, 3.75 pmol; ar-ketoglutarate, pH 7.9, 6 (I;mol; GABAase enzyme system (Sigma Chemical Co., Inc.), O-1 ml; and O-1 ml of the TGF-II samples. The reaction was initiated with the addition of tissue extract instead of u-ketoglutarate so that any endogenous cr-ketoglutarate in the extract would not start the reaction before the time desired; otherwise the procedures followed those recommended by the manufacturer. Absorbance at 340 nm was determined at intervals over a 60 min period and the GABA concentration of extracts estimated by comparison to a standard curve. RESULTS
Bean leaves inoculated with A. tumfuciens strain ATCC 13333 (Table 1) produce a substance that is extracted with phosphate buffer and promotes growth of strain B6 TABLE 1
phosphate buffer (TGF-II) and aqueous methanol Tumor growth-promoting activity of successive (TGF-I) extracts of control and ATCC 13333 inoculated leaf tissue Additions to bean leaves
No. of leaves with tumors
Mean tumor no. per leaf (range)
No. of tumors measured
Mean tumor diameter (S.U.)”
Change in tumor diameter
HsO control
16
28.2 (12-49)
47
5.7+0*1
-
Camosine, 1 m M
16
24.2
45
6.6kO.2
4-0.9
25.4 (12-41) 17.8 (7-46) 28.9 (1343) 20.1 (9-39)
42 45 47 37
5.8kO.l 5*5*0*1 5.6kO.l 56&-0.1
so.1 -0.2 -0.1 -0.1
Extracts of 13333~inoculated leaves TGF-II first 14 19.6 (4-35) TGF-I second 16 35.5 (19-78) TGF-I tist 16 22.3 (1042) TGF-II second 16 26.5 (9-61)
39 47 47 45
6.7kO.2 5.8+0-l 5.9kO.2 6.4AO.2
+1.0 i-o.1 +0*2 +0*7
Extracts of control leaves TGF-II first 14 TGF-I second 16 TGF-I first 16 TGF-II second 14
(446)
a s.u . = microscope ocular scale units.
One S.U. = O-033 mm.
tumors (tumor growth factor II or TGF-II). A previously described tumor growth factor (TGF-I) which is extracted with aqueous methanol [14] was not detected in ATCC 13333~inoculated leaves. The aqueous methanol TGF-I extraction procedure, therefore, must not extract TGF-II. Neither TGF-I nor TGF-II
334
K. E. Peters and J. A. Lippincott
activity was detected by bioassay of extracts from control leaves following these procedures. Comparison of the tumor growth-promoting activity of phosphate buffer extracts from leaves inoculated with other strains of Agrobacterium (Table 2) showed that strain 181 inoculated leaves do not produce TGF-II. The initial phosphate buffer TABLE 2
Comparative activity of phosphate buffer (TGF-II) and aqueous methanol (TGF-I) leaves inoculakd with dz&ent strains of Agrobacterium Source of extracts
-
Additions to BG-inoculated leave@ Ha0 control
extracts from
No. of tumors measured
Mean tumor diameter (S.U.)b
Change in tumor diameter
24
5.6kO.l
-
Carnosine, 1 rn~
28
6.2 + 0.2
+0.6
Control leaf
TGF-II extract TGF-I extract
34 21
5.7kO.l 5.550.2
+0.1 -0.1
ATCC 13333inoculated leaves
TGF-II extract TGF-I extract
34 28
6.4kO.2 5.5kO.l
+0.8 -0.1
ATCC 15834inoculated leaves
TGF-II extract TGF-I extract
28 30
6.1 +O.l 5.6kO.2
+0.5 0.0
181-inoculated leaves BG-inoculated leaves
TGF-II extract TGF-I extract TGF-II extract TGF-I extract
27 18 34 30
5.6kO.l 6.8kO.2 5.9+0*1 6.220.2
0.0 i- 1.2 +0.3 +0.6
ATCC 15955inoculated leaves
TGF-II extract TGF-I extract
28 27
6.0 + 0.2 6.3 + 0.2
+ 0*7c + l.OC
a TGF-II extracts made first, the residues were then extracted for TGF-I. b See Table 1. c These values from a separate experiment where the mean tumor diameter control tumors was 5.3 * 0.1.
of the
extract showed no growth-promoting activity; a subsequent aqueous methanol extract yielded TGF-I activity. Since growth-promoting activity was detected in both types of extracts from leaves inoculated with strains ATCC 15955 and B6, these bacteria induce both TGF-I and TGF-II formation. A. rhizogenes strain ATCC 15834, like strain ATCC 13333, has a low specific infectivity on bean leaves [12]. Similarly, it also induces TGF-II production in inoculated leaves but no detectable TGF-I. Only strain 181, of the 28 strains of Agrobacterium tested, did not induce TGF-II formation (Table 3). However, all other virulent strains, as well as four avirulent strains, induced TGF-II formation. Several virulent strains failed to induce TGF-I formation. There was no correlation between TGF-II production and the ability of the bacteria to utilize octopine or nopaline and hence to induce octopine- or nopalineproducing tumors [13, 201. The demonstration that TGF-II activity is due to GABA [17] permitted direct estimates of GABA levels in partly purified leaf extracts using an enzymatic assay [3]. TGF-II extracts from control and wounded leaves contained similar amounts of GABA, O-6 to O-7 pg/g fresh weight. The level of GABA in TGF-II extracts of strain ATCC 13333-inoculated leaves was I.8 pg/g fresh weight or about 3-fold greater.
Crown-gall
tumors
335
and GABA TABLE 3
Abili&v of Agrobacterium
strains to induce TGF-I and TGF-II
Strains of bacteria used
TGF-II production
TGF-I production
Infectivity
to inoculate leaves
formation
A. tumefaGzhls 15955,4,4452, TT107, C A6, B6, B2, Wl T-37, C-58-3, H-100 13333,5 AT3, ATl, AT4 IIBNVG, B-48 181
+ + -I-
-I+ + + +
-I-I-
+
A. rhizogem T‘R107, TR7 15834, 11325, TRlOl
+ -I-
A. zadiobacter TRl 4718 A. rubi 13334
+ + -l+ + + -
+
+
-I-l-
-I-
+ +
?
+
The usual TGF-II extracts (Tables 1 and 2) contain the equivalent of 10 g leaf tissue per ml and in the case of strain ATCC 133334nocuIated leaves the concentration of GABA in the extracts can be estimated to be c. l-7 x IO4 M, and in control and wounded leaves to be c. O-6 x 10-P M. Significant growth effects on tumors induced by strain B6 were obtained with GABA concentrations below 10V6 M (Fig. l), indicating that the tumors are sensitive to concentrations of GABA which are lOOO-fold lower than octopine. Since only O-1 ml of solution is applied per leaf, less than 10 ng of GABA per leaf is capable of eliciting a growth response. There is a clear saturation of the growth response to GABA which in most experiments occurs at concentrations of 10m5M and above.
~-Control I
I lo-’
I 10-e
I 10-5
I
I
10-4
10-3
I 10-Z
Concentration applied (M) FIG. 1. Growth of tumors in response to different concentrations Standard errors for the values shown were all within + O-2 S.U. 28
of GABA or octopiue.
K. E. Peters and J. A. Lippincott
886
The growth response to GABA is quite specific (Table 4) ; both u- and p-aminobutyric acids and a, ,&diaminobutyric acid are inactive in the bioassay. Only y-hydroxy-p-aminobutyrate, of the related amino acids tested, showed significant activity. TABLE 4
Tumor growth-promoting activity of GABA and related compounds Additions to BG-inoculated leaves
No. of tumors
Experiment la HsO control
Change in tumor diameter
-
5.2 + 0.2 5.5kO.2 5.2kO.2 5.2kO.l 5.3kO.l 5*3+0*1 5.4 + o-2 5.8kO.2
f0.3 0.0 o-o +0*1 +0.1 + 0.2 +0.6
38 25
6.1kO.2 7.0 + 0.2
$0.9
32
7.550.2
+1-4
Octopine n-a-Amino-n-butyrate r.-a-Amino-n-butyrate D, L+Amino-n-butyrate D, L-Y, P-Diaminobutyrate D, L-y-Amino-3-hydroxybutyrate y-Aminobutyrate (GABA) Experiment 2 HsO control y-Hydroxy-p-aminobutyrate
Mean tumor diameter (S.U.)b
(GOBAB) (1 mM) ykninobutyrate
(GABA)
(0.03 mM)
a Applied at 1 mu concentration b See Table 1.
except as indicated.
Despite its effects on tumor growth when added at day 3, the addition of GABA (IO-’ to lOa M) to leaves 15 min after inoculation does not promote tumor initiation. At 10e5 M GABA the number oftumors initiated is reduced by about 30% and 10e3 M GABA reduces tumor number by about 70%. DISCUSSION A. tumefaciens strain ATCC 13333, which induces only TGF-II, and strain 181, which induces only TGF-I, have allowed the demonstration that these factors can be differentially and sequentially extracted from the same tissue. TGF-II activity determined in extracts from leaves inoculated with 28 different strains of Agrobacterium shows that only strain 181 lacks the ability to induce TGF-II formation. Strain 181, Several non-tumorigenic strains also induce nevertheless, is highly tumorigenic. TGF-II activity, just as certain avirulent strains were shown previously to induce TGF-I formation [II]. Neither growth factor, therefore, can be considered essential for tumor development or to determine tumorous growth. Rather, they represent accessory changes associated with tumor initiation by particular strains of Agrobacterium. When these changes occur in the infection, however, they are important for growth of bean leaf tumors in vivo. Comparison of the ability of different bacteria to induce TGF-II, TGF-I, octopine or nopaline shows TGF-II formation to be the most typical change caused by Agrobacterium infection. The lack of TGF-II induction by strain 181 correlates with
Crown-gall tumors and GABA
337
another unique feature of the tumors induced by this strain: they produce neither octopine nor nopaline. Thus, among tumorigenic strains there may be a causal relation between the induction of TGF-II formation and production of one of these unusual guanidines. Because TGF-II or GABA activity was not detected in control leaf extracts by bioassay, it is tempting to suggest the above results are indicative of major increases in GABA levels in infected leaves. The S-fold increase in GABA in extracts from infected leaves is consistent with this possibility. However, the amount of GABA in control extracts and the high sensitivity of tumor growth to GABA (Fig. 1) clearly indicate that control extracts should produce a growth response. Because they do not, though they contain sufficient GABA, we postulate the existence of a second substance in control leaf extracts which interferes with the GABA-induced growth promotion. A decrease in the level of this hypothetical substance as opposed to change only in the level of GABA, consequently may account for much of the difference in activity of the control and tumorous leaf extracts. GABA is a common constituent of both plants and animals where its formation from glutamate and conversion to succinate provides an important shunt to the citric acid cycle [3, 251. Corn seedlings inoculated with the corn smut fungus, U&ago maydis, showed increased levels of GABA [19]. Thus, the hypertrophy associated with this disease may also involve changes in GABA metabolism. Steward et al. [24] found GABA levels increased in proliferating potato tuber tissue in culture as compared to resting tissue, whereas the content of other amino acids decreased. Promotion of plant growth by exogenous GABA has been noted by several investigators [2, 9, 18, 211. The GABA shunt also appears to be important in the germination of seeds [1, 8, 221, pollen [9] and bacterial spores [7]. Meins [15] showed that the metabolism of glutamate is important in the regulation of various morphogenic forms of a teratoma strain of cultured crown-gall tumor. Glutamic acid levels increase up to 3-fold in Agrobacterium inoculated tissues over controls during the period of tumor initiation [4] which could portend an increase in GABA levels. Srivastava & Naik [23] f ound the level of glutamate decarboxylase to be higher in cultured crown-gall tumors from Rumex acetosa as compared to normal tissue and the level of GABA transaminase to be lower. GABA levels in the two cultured tissues, however, were similar. The sensitivity of tumor growth to low levels of GABA shows that simple nutritional supply cannot account for this response. The specificity of the growth effect for only GABA and y-hydroxy-p-aminobutyric acid (GOBAB) of several very similar compounds also argues for a unique regulatory effect of GABA. These two compounds are active on animal nerve tissues [5], whereas closely related compounds are not, and GOBAB also promotes wound healing in animals [16]. In animals, GABA acts as a transmitter in inhibitory neurons by altering their permeability to certain ions [.5]. Wood & Braun [26] have shown that cultured crown-gall tumor tissues exhibit a greater capacity to accumulate potassium than corresponding normal tissues and that high levels of salts can partly overcome the nutritional requirements of normal tissue which distinguish it from tumor tissue. Thus, changes in GABA levels in these tumors may contribute to differences in ion balance which are critical for maintenance of maximum tumor growth rate.
338
K. E. Peters and J. A. Lippincott
This investigation was supported by Grant No. CA05387, awarded by the National Cancer Institute, DHEW. The authors thank Professor Nagai of Nihon University School of Dentistry for a supply of GOBAB. REFERENCES 1. AND&S, I., GONZALES,P. & SANTOS-Runs, A. (1973). The presence and the influence of germination on the y-aminobutyrate pathway in Lupinus albus seeds. Physiological Chemishy and Physics 5,357-364. 2. BALL, E. (1960). Sterile culture of the shoot apex of Lu@ts albus. Growth 24, 91-l 10. 3. BAXTER, C. F. (1972). Assay of y-aminobutyric acid and enzymes involved in its metabolism. Methods in Neurochemistry 3, l-73. 4. BEHKI, R. M. & LESLEY, S. M. (1968). Free amino acids of sunflower hypocotyl tissue during crown-gall tumor development. Canadian Journal of Botany 46, 2 17-223. 5. CURTIS, D. R. & JOHNSTON,G. A. R. (1974). Ammo acid transmitters in the mammalian central nervous system, Reviews of Physiology 69,97-188. 6. EL KHALIFA, M. D. & LIPPINCO~ J. A. (1968). Extraction, assay and partial purification of a factor from tumorous leaves that promotes crown-gall tumor growth. Physiologiu plantarum 21, 742-753. 7. FOERSTER,C. W. & FOERSTER,H. F. (1973). Glutamic acid decarboxylase in spores of Bacillus megatizmiandits possible involvementinspore germination. Journalof Bac&rio1ogy114,1090-1098. 8. INATOMI, K. & SLAUGHTER,J. C. (1971). The role of glutamate decarboxylase and y-aminobutyric acid in germinating barley. Journal of Ex&rimental Botany 22, 561-571. 9. KENDALL, W. A., LOWE, R. H. & TAYLOR, N. L. (1971). Growth of red clover pollen. III. Free amino acid composition in grams and supplements to culture media. Crop S&ace 11, 112-l 14. 10. LIPPINCOTT, B. B. & LIPPINCOTT,J. A. (1970). Bioassay and attributes of a growth factor associated with crown-gall tumors. Plant Physiology 46, 708-714. 11. LIPPINCOTT, J. A. & LIPPINCOTT, B. B. (1969). T umor growth complementation among strains of Agrobacterium. Journal of Bacteriology 99,496-502. 12. LIPPINCOTT, J. A. & LIPPINCOTT, B. B. (1970). Enhanced tumor initiation by mixtures of tumorigenie and non-tumorigenic strains of Agrobacterium. Infection aad Immunity 2, 623-630. 13. LIPPINCO~, J. A. & LIPPINCOTI‘, B. B. (1975). The genus Agrobactetium and plant tumorigenesis. Annual Review of Mkrobiology 29, 377-405. 14. LIPPINCOTT, J. A., LIPPINCOTT, B. B. & CHANG, C.-C. (1972). Promotion of crown-gall tumor growth by lysopine, octopine, nopalme and carnosine. Plant Physiology 49, 131-137. 15. MEINS, F., JR. (1971). Regulation ofphenotypic expression in crown-gall teratoma tissuesoftobacco. Developmental Biologv 24, 287-300. 16. NAGAI, K. & SUGNAMA, H. (1972). Kl inische Wirksamkeit von 4-Hydroxy-3-amino-ButtersPure (GOBAB) bei Dekubitus (ErklZirung eines neuren Begriffes von Antiinflammation). Journal of the .Nihon Universi~ School of Dentistry 14, 53-57. 17. PETERS,K. E., LIPPINCO~, J. A. & STUDIER, M. (1974). Identification of a crown-gall tumor growth factor as GABA. Phytochemistiy 13, 2383-2386. 18. PETERSON,C. M. (1973). Nutritional requirements for ovule formation in excised pistils of .Nigella. AmericanJournal of Botany 60,381-386. 19. PeTHij, M. (1972). Change of free ammo acid content in corn tissues infected with corn smut (Ustilago maydis (DC.) Cda.) in the course of pathogenesis. Acta plpk$athologica academiae scientiarum hungaricae 7, 47-55. 20. PETIT, A., DELHAYE, S., TEMPL?,J. & MOREL, G. (1970). Recherches sur les guanidines des tissus de crown-gall. Mise en evidence d’une relation biochemique sptcifique entre les souches d’Agroba&rium tumftiens et les tumeurs qu’elles induisent. Physiologic Vkgkale 8, 205-312. 21. RIJVEN, A. H. G. C. (1960). On the utilization ofy-aminobutyric acid by wheat seedlings. Australian 3ournal of Biological Sciences13, 132-141. 22. SPEDDING, D. J. & WILSON, A. T. (1968). Studies of the early reactions in the germination of Sinapis alba seeds. Phytochemistry7,897-901. 23. SRIVASTAVA, S. K. & NAIK, B. I. (1971). Arginine metabolism in normal and tumor tissues of plants : Part I-A comparative study. Indian Journal of Biochemistry and Biofhytis 8, 195-l 96. 24. STEWARD, F. Cl., THOMPSON,J. F. & POLLARD, J. K. (1958). Contrasts in the nitrogenous composition of rapidly growing and non-growing plant tissues. Journal of Experimental Botany 9, l-10. 25. STREETER,J. G. & THOMPSON,J. F. (1972). In vivo and in vitro studies on y-aminobutyric acid metabolism with the radish plant (Raphanus sativus L.). Plant Physiolop 49, 579-584. 26. WOOD, H. N. & BRAUN, A. (1965). Studies on the net uptake of solutes by normal and crown-gall tumor cells, Proceedingsof the National Academy of Sciences54, 1532-l 538.
Formation of a growth factor (GABA) by Agrobacferium inoculated leaves and the effect of GABA on tumor initiation and growth K. E. PETERS-~and J. A. LIPPINCOTT Department of Biological Sciences,Northwestern University, Evanston, Illinois 60201, U.S.A. (Acceptedfor publication September1976)
Twenty-seven strains of Agrobacterium, including virulent and avirulent forms, when inoculated on bean leaves induce formation of tumor growth factor II (TGF-II) which has been shown to be y-aminobutyric acid (GABA). Only A. tumefacienrstrain 181 did not; since this strain is highly virulent, formation of TGF-II is not essential for tumor initiation. The level of GABA in bacteria-inoculated leaves is increased about S-fold over wounded controls. Bean leaf tumors show a positive growth response when as little as 0.1 pg of GABA is applied per leaf and this response shows the same high specificity found for animal neuron responses to GABA. Because of the sensitivity of the tumor growth assay and the failure to detect TGF-II activity in extracts of control leaves, it is postulated that a second factor which inhibits tumor response to GABA is present in these extracts but reduced in amount or absent in extracts from bacteriainoculated leaves. Addition of GABA with the bacterial inoculum inhibits tumor initiation.
INTRODUCTION
The isolation of a new crown-gall tumor growth factor (TGF-II) from tumors induced on primary bean leaves by Agrobacterium tumefaciensstrain 13333 was described and evidence presented for its identification as y-aminobutyric acid (GABA) [I?]. This compound may thus be added to a limited and unique group of non-protein amino acids, including octopine and nopaline, which are capable of promoting the growth of bean leaf tumors in uiuo [12, 141. This paper: (a) relates the induction of TGF-II by various strains of Agrobacterium to their virulence and their ability to induce formation of other growth factors and (b) further characterizes the growthpromoting effects of GABA on these tumors. MATERIALS
AND
METHODS
Growth and inoculation of bacteria All strains of Agrobacterium employed in these studies were grown to stationary phase in liquid media as previously described [12]. Primary bean leaves (Phaseolusvulgaris L. var. “Pinto”) were inoculated either in viuo or in vitro, each procedure yielding similar rest&s in terms of the tumor growth activity detected in extracts. The in vivo method of inoculation has been described elsewhere 1121. Briefly, the leaves of t Present address: Department California 91010, U.S.A.
of Medical Genetics, City of Hope National Medical Ccntre, Duarte,
332
K. E. Peters and J. A. Lippincott
young seedlings were spread with O-1 ml of a bacterial suspension (c. 9 x IO* organisms) in the presence of no. 400 grit carborundum by means of a glass rod. The inoculated leaves were harvested 6 to 7 days later for extraction. The in vitro procedures developed by Lippincott & Lippincott (unpublished data) used leaves from 7-day-old seedlings. However, they were removed from the plant, weighed and 10 to 25 g placed in a 15 cm diameter Petri dish. Bacterial inocula (1 ml of stationary phase culture containing c. 9 x lOa bacteria per g of tissue) were added to the dish and the leaves chopped with a razor blade in the presence of the inoculum to yield leaf segments averaging G. 2 x 2 mm. The chopped tissue was then loosely packed in a 250 ml beaker, covered with aluminum foil, and incubated at 27 “C for 18 to 24 h. At this time the tissue was extracted or placed at -20 “C for later extraction. Bacteria for the in vitro inoculations were sedimented by centrifugation at 12 000 g for 20 min, resuspended in 10 mM sodium phosphate buffer, pH 7, and the procedure repeated. The washed bacteria were resuspended in phosphate buffer at their original concentration for inoculation of leaves by chopping. Preparation of extracts Tumor growth factor I (TGF-I), which is believed to be a derivative of histidine [la], was extracted by grinding inoculated leaves in aqueous methanol as described by El Khalifa & Lippincott [6J. Similar procedures were used to extract TGF-II except that 10 mM sodium phosphate buffer, pH 7, was used as the extracting medium [I7]. The extracts were pressed through four layers of cheesecloth, centrifuged at 10 000 g for 15 min at 4 “C. The supernatants containing either TGF-I or TGF-II were then filtered through Whatman no. 1 filter paper and evaporated to dryness in vacuaat 40 “C and redissolved in 1 ml of water per g of initial leaf tissue. To determine the presence of both TGF-I and TGF-II in the same tissue, two extractions were performed in succession using the different solvents: i.e. the residue from the first extraction retained by the cheesecloth plus the precipitate obtained by the initial centrifugation were combined and re-extracted with a second solvent. The order of extraction had no significant qualitative or quantitative effect on the activity detected in the resultant extracts. Tumor growth bioassay The activity of TGF-I, TGF-II and GABA was determined in viva using the bean leaf tumor growth bioassay described by Lippincott & Lippincott [JO]. These data are presented as the mean diameter of the tumors measured for each sample + the standard error of the mean. Where the standard errors are 3- 0.2 S.U. (see Table 1) differences between experimental and control values of O-4 S.U. are significant at the P=O*O5 level and differences of 0.6 S.U. are significant at the P=O*Ol level. Estimation of GABA levels The amount of GABA in leaf extracts was estimated by following NADP reduction in a coupled enzymatic reaction involving deamination of GABA and oxidation of the resultant succinic semi-aldehyde to succinate, This assay is specific for GABA [3]. Prior to assay, the crude TGF-II extracts were partitioned 3 times with ethyl acetate
333
Crown-gall tumors and GABA
and the organic phase containing TGF-II was evaporated to dryness and re-dissolved in water. The TGF-II was then streaked on a preparative silica gel thin-layer chromatography plate (Merck no. 5766) and developed with n-butanol-acetic acid-water (4 : 1 : 1). The silica gel at the R, of GABA was then scraped from the plate and eluted 3 times with water. The eluates were combined, evaporated to dryness and re-dissolved in 1 ml of water. One-tenth ml aliquots, representing c. 2 g initial leaf tissue, were used in the enzymatic assay. The reaction mixture contained the following reagents in a total volume of 3 ml: pyrophosphate buffer, pH 8.1, O-6 mol; 2-mercaptoethanol, 0*01.5 mmol; NADP (sodium salt) pH 7.9, 3.75 pmol; ar-ketoglutarate, pH 7.9, 6 (I;mol; GABAase enzyme system (Sigma Chemical Co., Inc.), O-1 ml; and O-1 ml of the TGF-II samples. The reaction was initiated with the addition of tissue extract instead of u-ketoglutarate so that any endogenous cr-ketoglutarate in the extract would not start the reaction before the time desired; otherwise the procedures followed those recommended by the manufacturer. Absorbance at 340 nm was determined at intervals over a 60 min period and the GABA concentration of extracts estimated by comparison to a standard curve. RESULTS
Bean leaves inoculated with A. tumfuciens strain ATCC 13333 (Table 1) produce a substance that is extracted with phosphate buffer and promotes growth of strain B6 TABLE 1
phosphate buffer (TGF-II) and aqueous methanol Tumor growth-promoting activity of successive (TGF-I) extracts of control and ATCC 13333 inoculated leaf tissue Additions to bean leaves
No. of leaves with tumors
Mean tumor no. per leaf (range)
No. of tumors measured
Mean tumor diameter (S.U.)”
Change in tumor diameter
HsO control
16
28.2 (12-49)
47
5.7+0*1
-
Camosine, 1 m M
16
24.2
45
6.6kO.2
4-0.9
25.4 (12-41) 17.8 (7-46) 28.9 (1343) 20.1 (9-39)
42 45 47 37
5.8kO.l 5*5*0*1 5.6kO.l 56&-0.1
so.1 -0.2 -0.1 -0.1
Extracts of 13333~inoculated leaves TGF-II first 14 19.6 (4-35) TGF-I second 16 35.5 (19-78) TGF-I tist 16 22.3 (1042) TGF-II second 16 26.5 (9-61)
39 47 47 45
6.7kO.2 5.8+0-l 5.9kO.2 6.4AO.2
+1.0 i-o.1 +0*2 +0*7
Extracts of control leaves TGF-II first 14 TGF-I second 16 TGF-I first 16 TGF-II second 14
(446)
a s.u . = microscope ocular scale units.
One S.U. = O-033 mm.
tumors (tumor growth factor II or TGF-II). A previously described tumor growth factor (TGF-I) which is extracted with aqueous methanol [14] was not detected in ATCC 13333~inoculated leaves. The aqueous methanol TGF-I extraction procedure, therefore, must not extract TGF-II. Neither TGF-I nor TGF-II
334
K. E. Peters and J. A. Lippincott
activity was detected by bioassay of extracts from control leaves following these procedures. Comparison of the tumor growth-promoting activity of phosphate buffer extracts from leaves inoculated with other strains of Agrobacterium (Table 2) showed that strain 181 inoculated leaves do not produce TGF-II. The initial phosphate buffer TABLE 2
Comparative activity of phosphate buffer (TGF-II) and aqueous methanol (TGF-I) leaves inoculakd with dz&ent strains of Agrobacterium Source of extracts
-
Additions to BG-inoculated leave@ Ha0 control
extracts from
No. of tumors measured
Mean tumor diameter (S.U.)b
Change in tumor diameter
24
5.6kO.l
-
Carnosine, 1 rn~
28
6.2 + 0.2
+0.6
Control leaf
TGF-II extract TGF-I extract
34 21
5.7kO.l 5.550.2
+0.1 -0.1
ATCC 13333inoculated leaves
TGF-II extract TGF-I extract
34 28
6.4kO.2 5.5kO.l
+0.8 -0.1
ATCC 15834inoculated leaves
TGF-II extract TGF-I extract
28 30
6.1 +O.l 5.6kO.2
+0.5 0.0
181-inoculated leaves BG-inoculated leaves
TGF-II extract TGF-I extract TGF-II extract TGF-I extract
27 18 34 30
5.6kO.l 6.8kO.2 5.9+0*1 6.220.2
0.0 i- 1.2 +0.3 +0.6
ATCC 15955inoculated leaves
TGF-II extract TGF-I extract
28 27
6.0 + 0.2 6.3 + 0.2
+ 0*7c + l.OC
a TGF-II extracts made first, the residues were then extracted for TGF-I. b See Table 1. c These values from a separate experiment where the mean tumor diameter control tumors was 5.3 * 0.1.
of the
extract showed no growth-promoting activity; a subsequent aqueous methanol extract yielded TGF-I activity. Since growth-promoting activity was detected in both types of extracts from leaves inoculated with strains ATCC 15955 and B6, these bacteria induce both TGF-I and TGF-II formation. A. rhizogenes strain ATCC 15834, like strain ATCC 13333, has a low specific infectivity on bean leaves [12]. Similarly, it also induces TGF-II production in inoculated leaves but no detectable TGF-I. Only strain 181, of the 28 strains of Agrobacterium tested, did not induce TGF-II formation (Table 3). However, all other virulent strains, as well as four avirulent strains, induced TGF-II formation. Several virulent strains failed to induce TGF-I formation. There was no correlation between TGF-II production and the ability of the bacteria to utilize octopine or nopaline and hence to induce octopine- or nopalineproducing tumors [13, 201. The demonstration that TGF-II activity is due to GABA [17] permitted direct estimates of GABA levels in partly purified leaf extracts using an enzymatic assay [3]. TGF-II extracts from control and wounded leaves contained similar amounts of GABA, O-6 to O-7 pg/g fresh weight. The level of GABA in TGF-II extracts of strain ATCC 13333-inoculated leaves was I.8 pg/g fresh weight or about 3-fold greater.
Crown-gall
tumors
335
and GABA TABLE 3
Abili&v of Agrobacterium
strains to induce TGF-I and TGF-II
Strains of bacteria used
TGF-II production
TGF-I production
Infectivity
to inoculate leaves
formation
A. tumefaGzhls 15955,4,4452, TT107, C A6, B6, B2, Wl T-37, C-58-3, H-100 13333,5 AT3, ATl, AT4 IIBNVG, B-48 181
+ + -I-
-I+ + + +
-I-I-
+
A. rhizogem T‘R107, TR7 15834, 11325, TRlOl
+ -I-
A. zadiobacter TRl 4718 A. rubi 13334
+ + -l+ + + -
+
+
-I-l-
-I-
+ +
?
+
The usual TGF-II extracts (Tables 1 and 2) contain the equivalent of 10 g leaf tissue per ml and in the case of strain ATCC 133334nocuIated leaves the concentration of GABA in the extracts can be estimated to be c. l-7 x IO4 M, and in control and wounded leaves to be c. O-6 x 10-P M. Significant growth effects on tumors induced by strain B6 were obtained with GABA concentrations below 10V6 M (Fig. l), indicating that the tumors are sensitive to concentrations of GABA which are lOOO-fold lower than octopine. Since only O-1 ml of solution is applied per leaf, less than 10 ng of GABA per leaf is capable of eliciting a growth response. There is a clear saturation of the growth response to GABA which in most experiments occurs at concentrations of 10m5M and above.
~-Control I
I lo-’
I 10-e
I 10-5
I
I
10-4
10-3
I 10-Z
Concentration applied (M) FIG. 1. Growth of tumors in response to different concentrations Standard errors for the values shown were all within + O-2 S.U. 28
of GABA or octopiue.
K. E. Peters and J. A. Lippincott
886
The growth response to GABA is quite specific (Table 4) ; both u- and p-aminobutyric acids and a, ,&diaminobutyric acid are inactive in the bioassay. Only y-hydroxy-p-aminobutyrate, of the related amino acids tested, showed significant activity. TABLE 4
Tumor growth-promoting activity of GABA and related compounds Additions to BG-inoculated leaves
No. of tumors
Experiment la HsO control
Change in tumor diameter
-
5.2 + 0.2 5.5kO.2 5.2kO.2 5.2kO.l 5.3kO.l 5*3+0*1 5.4 + o-2 5.8kO.2
f0.3 0.0 o-o +0*1 +0.1 + 0.2 +0.6
38 25
6.1kO.2 7.0 + 0.2
$0.9
32
7.550.2
+1-4
Octopine n-a-Amino-n-butyrate r.-a-Amino-n-butyrate D, L+Amino-n-butyrate D, L-Y, P-Diaminobutyrate D, L-y-Amino-3-hydroxybutyrate y-Aminobutyrate (GABA) Experiment 2 HsO control y-Hydroxy-p-aminobutyrate
Mean tumor diameter (S.U.)b
(GOBAB) (1 mM) ykninobutyrate
(GABA)
(0.03 mM)
a Applied at 1 mu concentration b See Table 1.
except as indicated.
Despite its effects on tumor growth when added at day 3, the addition of GABA (IO-’ to lOa M) to leaves 15 min after inoculation does not promote tumor initiation. At 10e5 M GABA the number oftumors initiated is reduced by about 30% and 10e3 M GABA reduces tumor number by about 70%. DISCUSSION A. tumefaciens strain ATCC 13333, which induces only TGF-II, and strain 181, which induces only TGF-I, have allowed the demonstration that these factors can be differentially and sequentially extracted from the same tissue. TGF-II activity determined in extracts from leaves inoculated with 28 different strains of Agrobacterium shows that only strain 181 lacks the ability to induce TGF-II formation. Strain 181, Several non-tumorigenic strains also induce nevertheless, is highly tumorigenic. TGF-II activity, just as certain avirulent strains were shown previously to induce TGF-I formation [II]. Neither growth factor, therefore, can be considered essential for tumor development or to determine tumorous growth. Rather, they represent accessory changes associated with tumor initiation by particular strains of Agrobacterium. When these changes occur in the infection, however, they are important for growth of bean leaf tumors in vivo. Comparison of the ability of different bacteria to induce TGF-II, TGF-I, octopine or nopaline shows TGF-II formation to be the most typical change caused by Agrobacterium infection. The lack of TGF-II induction by strain 181 correlates with
Crown-gall tumors and GABA
337
another unique feature of the tumors induced by this strain: they produce neither octopine nor nopaline. Thus, among tumorigenic strains there may be a causal relation between the induction of TGF-II formation and production of one of these unusual guanidines. Because TGF-II or GABA activity was not detected in control leaf extracts by bioassay, it is tempting to suggest the above results are indicative of major increases in GABA levels in infected leaves. The S-fold increase in GABA in extracts from infected leaves is consistent with this possibility. However, the amount of GABA in control extracts and the high sensitivity of tumor growth to GABA (Fig. 1) clearly indicate that control extracts should produce a growth response. Because they do not, though they contain sufficient GABA, we postulate the existence of a second substance in control leaf extracts which interferes with the GABA-induced growth promotion. A decrease in the level of this hypothetical substance as opposed to change only in the level of GABA, consequently may account for much of the difference in activity of the control and tumorous leaf extracts. GABA is a common constituent of both plants and animals where its formation from glutamate and conversion to succinate provides an important shunt to the citric acid cycle [3, 251. Corn seedlings inoculated with the corn smut fungus, U&ago maydis, showed increased levels of GABA [19]. Thus, the hypertrophy associated with this disease may also involve changes in GABA metabolism. Steward et al. [24] found GABA levels increased in proliferating potato tuber tissue in culture as compared to resting tissue, whereas the content of other amino acids decreased. Promotion of plant growth by exogenous GABA has been noted by several investigators [2, 9, 18, 211. The GABA shunt also appears to be important in the germination of seeds [1, 8, 221, pollen [9] and bacterial spores [7]. Meins [15] showed that the metabolism of glutamate is important in the regulation of various morphogenic forms of a teratoma strain of cultured crown-gall tumor. Glutamic acid levels increase up to 3-fold in Agrobacterium inoculated tissues over controls during the period of tumor initiation [4] which could portend an increase in GABA levels. Srivastava & Naik [23] f ound the level of glutamate decarboxylase to be higher in cultured crown-gall tumors from Rumex acetosa as compared to normal tissue and the level of GABA transaminase to be lower. GABA levels in the two cultured tissues, however, were similar. The sensitivity of tumor growth to low levels of GABA shows that simple nutritional supply cannot account for this response. The specificity of the growth effect for only GABA and y-hydroxy-p-aminobutyric acid (GOBAB) of several very similar compounds also argues for a unique regulatory effect of GABA. These two compounds are active on animal nerve tissues [5], whereas closely related compounds are not, and GOBAB also promotes wound healing in animals [16]. In animals, GABA acts as a transmitter in inhibitory neurons by altering their permeability to certain ions [.5]. Wood & Braun [26] have shown that cultured crown-gall tumor tissues exhibit a greater capacity to accumulate potassium than corresponding normal tissues and that high levels of salts can partly overcome the nutritional requirements of normal tissue which distinguish it from tumor tissue. Thus, changes in GABA levels in these tumors may contribute to differences in ion balance which are critical for maintenance of maximum tumor growth rate.
338
K. E. Peters and J. A. Lippincott
This investigation was supported by Grant No. CA05387, awarded by the National Cancer Institute, DHEW. The authors thank Professor Nagai of Nihon University School of Dentistry for a supply of GOBAB. REFERENCES 1. AND&S, I., GONZALES,P. & SANTOS-Runs, A. (1973). The presence and the influence of germination on the y-aminobutyrate pathway in Lupinus albus seeds. Physiological Chemishy and Physics 5,357-364. 2. BALL, E. (1960). Sterile culture of the shoot apex of Lu@ts albus. Growth 24, 91-l 10. 3. BAXTER, C. F. (1972). Assay of y-aminobutyric acid and enzymes involved in its metabolism. Methods in Neurochemistry 3, l-73. 4. BEHKI, R. M. & LESLEY, S. M. (1968). Free amino acids of sunflower hypocotyl tissue during crown-gall tumor development. Canadian Journal of Botany 46, 2 17-223. 5. CURTIS, D. R. & JOHNSTON,G. A. R. (1974). Ammo acid transmitters in the mammalian central nervous system, Reviews of Physiology 69,97-188. 6. EL KHALIFA, M. D. & LIPPINCO~ J. A. (1968). Extraction, assay and partial purification of a factor from tumorous leaves that promotes crown-gall tumor growth. Physiologiu plantarum 21, 742-753. 7. FOERSTER,C. W. & FOERSTER,H. F. (1973). Glutamic acid decarboxylase in spores of Bacillus megatizmiandits possible involvementinspore germination. Journalof Bac&rio1ogy114,1090-1098. 8. INATOMI, K. & SLAUGHTER,J. C. (1971). The role of glutamate decarboxylase and y-aminobutyric acid in germinating barley. Journal of Ex&rimental Botany 22, 561-571. 9. KENDALL, W. A., LOWE, R. H. & TAYLOR, N. L. (1971). Growth of red clover pollen. III. Free amino acid composition in grams and supplements to culture media. Crop S&ace 11, 112-l 14. 10. LIPPINCOTT, B. B. & LIPPINCOTT,J. A. (1970). Bioassay and attributes of a growth factor associated with crown-gall tumors. Plant Physiology 46, 708-714. 11. LIPPINCOTT, J. A. & LIPPINCOTT, B. B. (1969). T umor growth complementation among strains of Agrobacterium. Journal of Bacteriology 99,496-502. 12. LIPPINCOTT, J. A. & LIPPINCOTT, B. B. (1970). Enhanced tumor initiation by mixtures of tumorigenie and non-tumorigenic strains of Agrobacterium. Infection aad Immunity 2, 623-630. 13. LIPPINCO~, J. A. & LIPPINCOTI‘, B. B. (1975). The genus Agrobactetium and plant tumorigenesis. Annual Review of Mkrobiology 29, 377-405. 14. LIPPINCOTT, J. A., LIPPINCOTT, B. B. & CHANG, C.-C. (1972). Promotion of crown-gall tumor growth by lysopine, octopine, nopalme and carnosine. Plant Physiology 49, 131-137. 15. MEINS, F., JR. (1971). Regulation ofphenotypic expression in crown-gall teratoma tissuesoftobacco. Developmental Biologv 24, 287-300. 16. NAGAI, K. & SUGNAMA, H. (1972). Kl inische Wirksamkeit von 4-Hydroxy-3-amino-ButtersPure (GOBAB) bei Dekubitus (ErklZirung eines neuren Begriffes von Antiinflammation). Journal of the .Nihon Universi~ School of Dentistry 14, 53-57. 17. PETERS,K. E., LIPPINCO~, J. A. & STUDIER, M. (1974). Identification of a crown-gall tumor growth factor as GABA. Phytochemistiy 13, 2383-2386. 18. PETERSON,C. M. (1973). Nutritional requirements for ovule formation in excised pistils of .Nigella. AmericanJournal of Botany 60,381-386. 19. PeTHij, M. (1972). Change of free ammo acid content in corn tissues infected with corn smut (Ustilago maydis (DC.) Cda.) in the course of pathogenesis. Acta plpk$athologica academiae scientiarum hungaricae 7, 47-55. 20. PETIT, A., DELHAYE, S., TEMPL?,J. & MOREL, G. (1970). Recherches sur les guanidines des tissus de crown-gall. Mise en evidence d’une relation biochemique sptcifique entre les souches d’Agroba&rium tumftiens et les tumeurs qu’elles induisent. Physiologic Vkgkale 8, 205-312. 21. RIJVEN, A. H. G. C. (1960). On the utilization ofy-aminobutyric acid by wheat seedlings. Australian 3ournal of Biological Sciences13, 132-141. 22. SPEDDING, D. J. & WILSON, A. T. (1968). Studies of the early reactions in the germination of Sinapis alba seeds. Phytochemistry7,897-901. 23. SRIVASTAVA, S. K. & NAIK, B. I. (1971). Arginine metabolism in normal and tumor tissues of plants : Part I-A comparative study. Indian Journal of Biochemistry and Biofhytis 8, 195-l 96. 24. STEWARD, F. Cl., THOMPSON,J. F. & POLLARD, J. K. (1958). Contrasts in the nitrogenous composition of rapidly growing and non-growing plant tissues. Journal of Experimental Botany 9, l-10. 25. STREETER,J. G. & THOMPSON,J. F. (1972). In vivo and in vitro studies on y-aminobutyric acid metabolism with the radish plant (Raphanus sativus L.). Plant Physiolop 49, 579-584. 26. WOOD, H. N. & BRAUN, A. (1965). Studies on the net uptake of solutes by normal and crown-gall tumor cells, Proceedingsof the National Academy of Sciences54, 1532-l 538.