Cytokinins: Production and Biogenesis of N6-(◿2-isopentenyl) Adenine in Cultures of Agrobacterium tumefaciens Strain B6

Botanisches Institut der Universitat Bonn

Cytokinins: Production and Biogenesis of N 6 -(Ll2-isopentenyl) Adenine in Cultures of Agrobacterium tumefaciens Strain B6 HEINZ HAHN, INGE HEITMANN

and

MARIANNE BLUMBACH

With 6 figures Received February 9, 1976· Accepted February 24, 1976

Summary The production of N6-(,d2-isopentenyl)adenine in Agrobacterium tume/aciens strain B6 grown in logarithmic phase was estimated to be 0.3 fig/I in the medium and 2.3 fig in the bacteria from 1 I medium. The cytokinin activity of tRNA isolated from the bacteria was estimated to be 10-9 mol cytokinin/mg tRNA after alkaline hydrolysis and 4.6 X 10-10 mol cytokinin/mg tRNA after enzymic hydrolysis. It was calculated that the amount of cytokinin released from tRNA of 2-3 g bacteria present in the medium at early logarithmic phase is sufficient to account for all the cytokinin found. This calculation is based on the estimated half-life of 27.5 hours for tRNA in A. tume/aciens and on the assumption that every tenth macromolecule does contain a cytokinin. The data show that tRNA can be considered as an intermediate in cytokinin biogenesis although the possibility can not be excluded that a second cytokinin biosynthetic pathway independent of tRNA may exist.

Key words: cytokinins, biogenesis, Agrobacterium tume/aciens.

Introduction

The plant pathogen Agrobacterium tumefaciens B6 is the causal organism of crown gall induction after wound infection in many plant species (LIPPINCOTT et aI., 1975). The enhanced cell division activitiy during tumor growth and, similarly, the growth of pea shoots after infection with Corynebacterium fascians, another plant pathogen, led to the assumption that these bacteria may give off substances which stimulate cell division. It could be shown that Corynebacterium produces N 6 -(,d2-isopentenyl) adenine (i 6 Ade), a potent cytokinin (KLAMBT et aI., 1966; HELGESON et aI., 1966). For A. tumefaciens B6 the isolation of material was reported with the same properties as i 6Ade (KLAMBT, 1967). Later UPPER et al. (1970) confirmed this result. As was shown by numerous investigations, i 6Ade occurs as integral part of tRNA in many organism (microorganisms, animals and plants) while as a free cytokinin (exhibiting cytokinin activity only in this form) it has been found in higher plants as well as some plant pathogens, parasites and symbiontes which cause symptoms in Z. P/lanzenphysiol. Bd. 79. S. 143-153. 1976.

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infected plants that can be mimicked by applied cytokinins (KENDE, 1971). The free cytokinin in the cell could be released on breakdown of tRNA making the molecule an intermediate in the biogenesis of cytokinin or it could be synthesized by an independent pathway (CHEN et aI., 1970). We set out to investigate these possibilities using the technique of half-life estimation for tRNA (LEINEWEBER et aI., 1974). But before we could proceed it was necessary to confirm the amount of free i6Ade found in the bacteria culture.

Materials and Methods 1. Bacteria culture For all investigations Agrobacterium tumefaciens strain B6 was used. The bacteria were grown in a defined liquid medium (MciNTIRE et aI., 1940) supplemented with double concentrations of the micronutrients reported by KLAMBT et al. (1966). The cultures were placed on a reciprocating shaker (67 oscillations/min) and grown at 27 DC in the dark to late logarithmic phase before harvesting by continuous-flow centrifugation at 15,000 g at 4 DC. The clear supernatant was used immediately. The bacteria were washed with 0.9 0/0 NaCI and stored at -20 DC until further use. 2. Isolation of cytokinin a) The supernatant was adjusted to pH 7.8 with sodium hydroxide and extracted four times with 1 volume of watersaturated ethyl acetate. The ethyl acetate was removed in a rotary evaporator at 40 DC, and the residue was taken up in a small volume of chloroformmethanol (7: 3, v/v) and chromatographed on a silica gel column (HAHN, 1975). The different fractions were pooled, reduced to a small volume and applied to system A: silica gel thin layer plates developed with chloroform-acetic acid (8: 2, v/v). The region of the cochromatographed authentic i6 Ade was scraped off and carefully eluted with 80 % ethanol. The eluted material was rechromatographed on silica gel plates in system B: chloroformmethanol (9 : 1, v/v). This procedure was repeated with system C: cellulose F thin layer plates developed with i-propanol-28 Ofo NH 4 0H-water (9 : 1 : 2, v/v). b) Frozen bacteria were allowed to soften at 4 DC and were then disrupted with aluminiumoxid in a chilled mortar kept on ice throughout the grinding procedure after LAMBORG (1967) using 95 % ethanol instead of buffer. The homogeneous suspension was stirred at 4 DC for 1 hour and then centrifuged at 25,000 g for 20 minutes. The supernatant was reduced to a small volume and subjected to TLC as described above. c) tRNA was extracted by a modified procedure of v. EHREN STEIN (1967) and VANDERHOEF et al. (1970). Bacteria were suspended in 2 volumes of buffer I (25 mM Tris-HCI, pH 7.5), then 4 volumes of phenol solution (500 g phenol, 70 ml m-cresol, 55 ml buffer I, C.5 g 8-hydroxyquinoline, KIRBY, 1965) were added and the resulting suspension shaken for 2 hours. All further operations were carried out at 4 DC unless specified otherwise. The resulting emulsion was separated by centrifugation (10,000 g). To the upper aqueous phase NaCI (30 mg/ml) and 0.5 volume phenol solution were added. The emulsion was shaken for 5 minutes, centrifuged and the aqueous phase collected. The bacteria suspension was reextracted and the aqueous phases pooled, 1/9 volume of 1.5 M K-acetate was added and the nucleic acids were precipitated with 2.5 volumes of ethanol-m-cresol (9 : 1, v/v). The sedimented nucleic acids were dissolved in buffer I and deproteinized twice with phenol solution and finally precipitated with cold ethanol. tRNA was extracted with 3 M NaZ. Pflanzenphysiol. Bd. 79. S. 143-153. 1976.

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acetate (VANDERHOEF et aI., 1970) and further purified on a DEAE-cellulose column (V. EHRENSTEIN, 1967). Gel electrophoresis of tRNA was carried out on 2.4 Ofo polyacrylamide gels (BISHOP et aI., 1967). The gels were scanned in a Gilford spectrophotometer. The amino acid acceptor assay of tRNA was performed by a procedure of CHERAYIL et a!. (1968) . The enzymic hydrolysis of tRNA and extraction of cytokinin ribosides followed the procedure of BURROWS et a!. (1969). Alkaline hydrolysis of tRNA was achieved after incubation with 0.3 M KOH for 18 hours at 37 °C. The hydrolysate was chilled and adjusted to pH 9 with 2.4 N perchloric acid. The insoluble material was centrifuged out and the resulting nucleosides extracted as described for the enzymic hydrolysis of tRNA. 3. Bioasssay The Funaria bioassay (HAHN et a!., 1968) was modified. The protonemata were cultured as described (STEGMANN et aI., 1974). Six days after onset of the culture most of the protonemata were removed with fine forceps leaving 6-8 well separated protonemata per dish. After 14-15 days the cellophane around the protonemata was cut with a razorblade and the protonemata with the underlying cellophane disk were transferred onto the test agar (3 protonemata per petri dish). After 40-48 hours the induced buds were counted under a microscope. Cytokinin activity is expressed as i 6 Ade or i 6Ado equivalents (i 6AdeE, i 6AdoE). 4. Crown gall test The pathogenicity of A. tume/aciens B6 was checked regularly by Its ability to induce crown gall in leaves of Kalanchoe daigremontiana (BEIDERBECK, 1970). 5. Hal/-life

0/ tRNA

The estimations were performed using 14C-orotic acid as described by LEINEWEBER et al. (1974) and KALMUTZKI (1975). 6. Cas liquid chromatography (CLC) GLC separation was performed in a model 419 Packard-Becker gas chromatograph fitted with flame ionization detectors. A 0.2 X 200 em coiled silanized glass column packed with 3 Ofo SE-30 on 80-100 mesh chromosorb W (HP) was conditioned 24 hours before use. The sample treatment for silylation was the same as described by HAHN (1975). The silylation procedure was performed as described by BUTTS (1970). 7. Chemicals Silica gel 60 for column chromatography 70-230 mesh, precoated silica gel 60 F254 and cellulose F thin layer plates were purchased from Merck; snake venom, alkaline phosphatase and aluminiumoxid Alcoa A-305 from Serva; 14C-orotic acid (61 mCi/ mmol), 14C-leucine (311 mCi/mmol) from Radiochemical Centre, Amersham; tRNA from Boehringer; i6 Ade from Waldhof, i6 Ado from Sigma; zeatin and zeatinriboside from Calbiochem; 3 Ofo SE-30 on 80-100 mesh chromosorb W (HP) from Pierce Chemical Compo

Results 1. Pathogenicity of A. tumefaciens

To be sure that the bacteria retained their ability to induce crown galls during the course of our experiments we tested them regularly. At the end of the investigations the bacteria showed their original pathogenicity (Table 1). Z. P/lanzenphysiol. Bd. 79. S. 143-153. 1976.

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Table 1: Development of crown gall at Kalanchoe daigremontiana after wound infection with different bacteria. Bacteria Agrobacterium tumefaciens B6':·) Corynebacterium fascians Escherichia coli buffer (BEIDERBECK, 1970)

I

Crown gall (¢ in mm) 2.8; 2.2; 2.0; 2.4; no crown gall induction

,:.) Each value is the average of 21 crown galls/leaf.

2. Cytokinin activity in the bacteria medium It has been reported that A. tumefaciens does produce 100 {lg i 6Ade/l bacteria medium (UPPER et aI., 1970). The production of a cytokinin in those large quantities by the bacteria made these organism especially suitable for our aim of elucidating the biogenesis of i6 Ade. For a rapid and reproducible quantitation of cytokinin in a large number of samples GLe was chosen. However in many experiments we could not confirm the data reported and what is more we were not able to indentify convincingly i6Ade in the medium by GLe. To test the sensitivity of our sytem (detection limit with authentic i6Ade at 5-10 ng) a test experiment was carried out. 150 {lg i 6Ade were dissolved in 1500 ml of water pH 7.5, extracted with ethyl acetate and the extract chromatographed in system A. Finally the appropriate zone was scraped off, eluted and prepared for GLe. The water phase (after ethyl acetate extraction) was reduced to dryness and the residue prepared for GLe. As expected the chromatogram of the ethyl acetate extract shows a distinct i 6 Ade peak, even 50

- i6 Ade

(a)

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.2 U

10

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.

"0

"0 0
:; I.L

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70 50

30 6

10

Retention time (min)

Fig. 1: GLC of an ethyl acetate extract from water containing 150 pg of i6Ade in 1500 ml of water (a) and the water residue (b). 1.1 pi (a) and 1.3 pI (b) of sample were injected. Flow rates (mllmin): N2 = 21, H2 = 45, air = 300. Injector and oven at 255 aC, flame ionization detector at 280 ac. Full scale deflection represents an ionization detector current of 3.3 X 10-9 (a) and 3.2 X 10-10 (b) amp at an attenuation of 1280 resp. 128.

z.

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though the sensitivity setting of the detector is low. In the water phase no i6Ade could be found with a setting ten times more sensitive (Fig. 1), thus confirming the complete extraction with ethyl acetate using i6 Ade-8- 14 C (HAHN, 1975). At this stage of our investigation it became clear that A. tumefaciens B6 must produce i6 Ade in an amount far less than reported. Therefore our aim was first to determine the amount of i6Ade using the Funaria bioassay which detects i6Ade up to 10-a M. The bacteria medium extracted with ethyl acetate was further purified using a standard silica gelcolumn (HAHN, 1975). The cytokinin activity eluted almost completely with fraction III (Fig. 2), cochromatographing mainly with authentic i6 Ade and i6 Ado. Little activity was found with fraction IV. Fraction III was further purified in system A, B and C always taking the zone which cochromatographed with authentic i6Ade and then bioassayed (Fig. 3). The cytokinin activity still cochromatographed with i6 Ade (Rf 0.79-0.85). Taking in consideration a recovery of 60 % (HAHN, 1975) when using the above procedure cytokinin activity was calculated to be 0.24 ,ug/ l i6 AdeE in the bacteria medium.

3. Cytokinin activity in the bacteria cells To determine the amount of the cytokinin inside the bacteria they were disrupted and then extracted with ethanol. The extract was purified in system A and B (taking E..

Froc;t.on

Fig. 2: Cytokinin activity in the effluent of a silica gel 60 column after chromatography with chloroform-methanol (7: 3, v / v). The column was equilibrated with eluent before applying the sample (4 ml) extracted from 61 bacteria medium. The different fraction were collected, purified on TLC as described under ~ Methods » and the cytokinin activity determined using the Funaria bioassay. Z. Pflanzenphysiol. Bd. 79. S. 143-153. 1976.

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200

150

100

50

o

0.5 RF

1.0

10'Y5xl0-910-tl5xl0' 10'7 j6 Ade 1M]

Fig. 3: Cytokinin activity of fraction III (s. Fig. 2) after TLC in system A, Band C. Bud induction by authentic i6 Ade is enclosed.

the zone which chochromathographed with authentic i6 Ade) and then bioassayed. (Cytokinin activity in system B, RF 0.24-0.32, authentic i6 Ade, RF 0.28-0.32). Taking into account the same recovery as above (600/0) we calculated 2.3 ~g117 g bacteria i6 Ade (1 I of a bacteria culture under our growth condtion yielded ca. 17 g bacteria). It should be mentioned that this value is in close agreement with cytokinin determination usmg high performance liquid chromatography (HAHN, m preparation).

4. Cytokinin activity in tRN A from A. tumefaciens B6 The tRNA obtained at this stage was of high spectral purity with a ratio of A 260 i280 = 2.1 and an A 260i230 = 2.2, and it was free of high molecular RNA (Fig. 4). The tRNA sample accepted 1 pmol of 14C-Ieucine/5 A 260 units under the assay condition used. Samples of tRNA subjected to either to enzymic or alkaline hydrolysis with subsequent treatment of phosphatase were further purified as Z. PJlanzenphysiol. Bd. 79. S. 143-153. 1976.

Production and biogenesis of the cyto~inin i6Ade

149

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Fig. 4: Electrophoresis of tRNA isolated from A . tumefaciens on 2.4 Ofo polyacrylamide gels. (A) after extraction with 3 M Na-acetate from total RNA (B). For comparison tRNA from Boehringer (C).

described and finally the riboside mixture was bioassayed. From these determinations we calculated the cytokinin yield of the isolated sRNA to be of 10- 9 mol i6AdoE after alkaline hydrolysis and 4.6 X 10-10 mol i6AdoE/mg sRNA after enzymic hydrolysis. The results show that the cytokinin activity after purification on silica gel column and different TIC system was mostly i 6Ade. This cytokinin was found in the medium but mainly in the bacteria cells and in the tRNA, although in the latter case an accurate determination of the nature of the cytokinin(s) was not undertaken. Thus the biogenesis pathway for i6Ade may become clear: tRNA as an intermediate in the biogenesis of the free cytokinin (CHEN et al., 1970). To prove this hypothesis in a different way we therefore determined the life span of tRNA enabling us to calculate how much the macromolecule may contribute to the supply of free cytokinin (LEINEWEBER et al., 1974).

5. Half life (tt /2) of tRNA in A. tumefaciens B6 The bacteria grown in logarithmic phase were pulsed with 14C-orotic acid (0.01 mM) for 8 hours , chased with 12C-orotic acid (0.01 M) and then again kept in logarithmic phase by proper dilution of the medium. Small samples of bacteria were removed at the indicated times and tRNA extracted and purified as described. 6 hours after having finished the incubation period the bacteria are still incorporating the labelled precursor into tRNA until the maximum is attained at 27 hours after onset of Z. PJlanzenphysiol. Bd. 79. S. 143-153. 1976.

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Fig. 5: 14C-radioactivity of tRNA from A. tumefaciens during chase incubation. Half life (t1/ 2) is marked. The amount of medium removed containing the bacteria from which tRNA was extracted was 10 ml. To keep the bacteria in logarithmic growth during the chase incubation, the medium was diluted and the removed sample was increased in proportion to the dilution factor. Inset: growth (absorbence at 600 nm) during chase incubation. The data are plotted on a semilogarithmic scale.

the chase period (Fig. 5). As shown in the graph a half life of 27.5 hours for tRNA of A. tumefaciens B6 can be calculated. Discussion Our results show that A. tumefaciens B6 does produce 2.6 /lg/l i6AdeE (bacteria plus medium) grown in the logarithmic phase. This amount is in the same order of magnitude of the first estimation of 10 /lg in which bacteria and medium were extracted together (KUi.MBT, 1967). But our estimation is in disagreement with the finding of UPPER et al. (1970) who reported a yield of 100 /lg/l in the medium. The situation is similar for Corynebacterim fascians another plant pathogen for which 100 /lg/ l i 6Ade were estimated (KLAMBT et al., 1966). Later a yield of only 2 /lg/ l of i6Ade was reported. If an acidification step is included in the extraction procedure the yield increases to 12 /lg/l. This is due to the release of i6Ade from tRNA during the extraction procedure (RATHBONE et al., 1972). Recently SCARBROUGH et al. (1973) Z. Pflanzenphysiol. Ed. 79. S. 143-153. 1976.

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reported an amount of i 6Ade for Corynebacterium of 1.2 .ug/l. Whether the high cytokinin amount mentioned above stems partly from release of tRNA due to breakdown during the isolation procedure or whether the bacteria have changed during the course of the experiments we do not know. Both parameters were checked carefully in our experiments and were excluded right from the beginning. In any case it seems obvious that 10 .ug/l are an upper limit but an amount of 2-4 .ug is probably the normal yield for these bacteria. Concerning the two possibilities of biogenesis of free cytokinin (Fig. 6): to our knowledge this is the first time that free and bound cytokinin are determined together with the half life of tRNA in an organism. This set of determination does allow one to estimate how much of the cytokinin could be supplied via the degradation pathway. If one mg tRNA (MW 27,000) is hydrolyzed completely and if every tenth molecule does contain one cytokinin (PETERKOFSKY, 1968), one would

PURINE POOL bases.nucleosides ..);:;:;m~==:" nucleotides

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Common abbreviation

H

H

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cis-Z

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H

trans-Z

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CH3S- H

trans~2m~Z

cytokinin nucleosides: cytokinin bases plus ribose as R3 cytokinin nucleotides : cytokinin bases plus ribose~ 5'- phosphate as R3

Fig. 6: Cytokinin biogenesis scheme. Not included in the scheme are metabolic conversions like i 6Ade -+ Z, Z:;:::!; RZ, RZ -+ RDZ, RDZ -+ DZ. i6Ade = N 6-(LF-isopentenyl)adenine, j6Ado = N 6-(,d2-isopentenyl)adenosine, Z = zeatin, RZ = ribosyl zeatin and the corresponding CH 3S = 2-methylthio derivatives. DZ = dihydrozeatin, RDZ = ribosyldihydrozeatin, IPP = LJ2-isopentenyl pyrophosphate, MVA = mevalonic acid. Z. PJlanzenphysiol. Bd. 79. S. 143-153. 1976.

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calculate a theoretical yield of free cytokinin of 3.7 X 10- 9 mo1!1mg tRNA, which could be supplied via the degradation pathway (breakdown of tRNA) in the cell. This would mean that only 2-3 g of these bacteria present in the medium in the early logarithmic phase could produce the amount of cytokinin after one half life (tl/2 = 27.5 hours) shown by the following calculation: 2-3 g bacteria contain 120-180 A 260 units (1 g bacteria contains 60 A 260 units, data not shown). After tl/2 60-90 A 260 units are hydrolyzed equivalent to 2.7-4 mg tRNA (22 A 260 units tRNA = 1 mg tRNA) yielding 10-8 -1.5 X 10-8 mol cytokinin or 2.2-3.3 {lg of a cytokinin base like i6Ade. In summary this calculation is consistent with our experimental data. This would mean that the amount of cytokinin we found (2.6 {lg i6 Ade) can be supplied indeed by breakdown of tRNA. This calculation is based on the assumption that every cytokinin molecule found in the macromolecule would be conducted into the free cytokinin pool, i.e., no breakdown or recycling of the cytokinin occurs. Our data show that tRNA can be considered as an intermediate in the biogenesis of cytokinin. What is more all degradative enzymes can be found in the cell which are needed to digest the macromolecule what leads to free cytokinin (HALL, 1974). But it should be mentioned that there are still difficulties understanding the way in which a macromolecule involved in the protein biosynthesis could be connected in a regulatory manner to the hormone supply of the cell. Although early investigations to elucidate a second tRNA-independent biosynthetic pathway using an in vitro system were not successful (HAHN et al., 1971) we can not exclude this possibility. A recent note on this subject (CHEN et al., 1975) has raised once more the question on this subject. For clarification an appropriate in vitro system including the purified enzyme tRNA-isopentenyltransferase (HOLTZ et aI., 1975) may be a good test system for further work. We would like to thank Dr. R. BEIDERBECK, Botanisches Institut, Universitat Heidelberg, for a generous supply of Agrobacterium tumefaciens B6; Mrs. H. MOBIUS for technical assistance and Dr. R. HABERSTROH for his help in the preparation of the english manuscript.

References BEIDERBECK, R.: Z. Naturforsch. 25 b, 407 (1970). BISHOP, D. H. L., J. R. CLAYBROOK, and S. SPIEGELMAN: J. Mol. BioI. 26, 373 (1967). BURROWS, W. J., D. J. ARMSTRONG, F. SKOOG, S. M. HECHT, J. T. A. BOYLE, N. ]. LEONARD, and ]. OCCOLOWITZ: Biochemistry 8, 3071 (1969). BUTTS, W. c.: J. Chromatogr. Sci. 8, 474 (1970). CHERAYIL, ]. D., A. HAMPEL, and R. M. BOCK: In Meth. of Enzymol. XII B, 166 (1968). CHEN, C. M., and R. L. ECKERT: Plant Physiol. Supplement 56,2, 29 (1975). CHEN, C. M., and R. H. HALL: Phytochem. 8,1687 (1970). EHREN STEIN, v. G.: in Meth. of Enzymol. XII A, 588 (1967). HAHN, H.: Physiol. Plant. 34, 204 (1975). HAHN, H., and M. Bopp: Planta (Berl.) 83, 115 (1968). HAHN, H., H. KENDE, and R. DE ZACKS: In Plant Research 70, 77 MSU/AEC Plant Research Laboratory, East Lansing (1971).

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HALL, R. H.: Ann. Rev. Plant Physiol. 24, 415 (1973). HELGESON, J. P., and N. J. LEONARD: Proc. Nat. Acad. Sci. USA 56, 60 (1966). HOLTZ, J., and D. KLAMBT: Hoppe Seyler's Z. Physiol. Chern. 356,1459 (1975). KALMUTZKI, K.: Staatsexamensarbeit, Universitat Bonn (1975). KENDE, H.: Int. Rev. Cytol. 31, 301 (1971). KIRBY, K. S.: Biochem. J. 96, 266 (1965). KLAMBT, D.: Wiss. Z. Univ. Rostock, Math.-Naturwiss. Reihe 16, 623 (1967). KLAMBT, D., G. THIEs, and F. SKOOG: Proc. Nat. Acad. Sci. USA 56, 52 (1966). LAMBORG, M.: in Meth. of Enzymol. XII A, 527 (1967). LEINEWEBER, M., and D. KLAMBT: Physiol. Plant. 30, 327 (1974). LIPPINCOTT, J. A., and B. A. LIPPINCOTT: Ann. Rev. Microbiol. 29, 377 (1975). McINTIRE, F. c., W. H. PETERSON, and A. J. RIKER: J. Agr. Res. 61, 313 (1940). PETERKOFSKY, A.: Biochemistry 7, 472 (1968). RATHBONE, M. P., and R. H. HALL: Planta (Berl.) 108,93 (1972). SCARBROUGH, E., D. J. ARMSTRONG, F. SKOOG, C. R. FRIHART, and N. J. LEONARD: Proc. Nat. Acad. Sci. USA 70, 3825 (1973). STEGMANN, J., and H. HAHN: Z. Pflanzenphysiol. 74, 143 (1974). UPPER, C. D., J. P. HELGESON, J. P. KEMP, and C. J. SCHMIDT: Plant Physiol. 45, 543 (1970). VANDERHOEF, L. N., R. F. BOHANNON, and J. L. KEY: Phytochem. 9, 2291 (1970).

Dr. HEINZ HAHN, Botanisches Institut der Universitat, Meckenheimer Allee 170, D-5300 Bonn, Germany.

Z. Pflanzenphysiol. Bd. 79. S. 143-153. 1976.