Planta (1984)161:345-354

P l a n t a 9 Springer-Verlag 1984

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue Ian M. Scott and Roger Horgan Department of Botany and Microbiology, University College of Wales, Aberystwyth, Dyfed SY23 3DA, UK

Abstract. The cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using 2H2-1abelled cytokinin riboside 5'-monophosphates and 15N4-1abelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5'-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells. Key words: Crown-gall - Cytokinin in crown-gall - Mass spectrometry (cytokinins) - Nicotiana (cytokinins).

Introduction Crown-gall tumours are induced on many species of higher plants by the pathogenic bacterium Agrobacterium tumefaciens. Tumorigenesis is associated with the stable incorporation into the plant nuclear genome of bacterial T-DNA, a specific portion of the Ti plasmid carried by virulent agrobacteria (Willmitzer et al. 1980). While the growth of normal plant cells in vitro generally requires the presence of auxin and cytokiAbbreviations: DHZ=dihydrozeatin; DHZ7G=dihydrozeatin

7-glucoside; DHZMP = dihydrozeati/a 9-riboside 5'-monophosphate; DHZR=dihydrozeatin 9-riboside; GC-MS=coupled gas chromatography-mass spectrometry; HPLC=high-performance liquid chromatography; Z7G=zeatin 7-glucoside; Z9G=zeatin 9-glucoside; ZOG=zeatin O-glucoside; ZMP= zeatin 9-riboside 5'-monophosphate; ZR=zeatin 9-riboside; ZROG = zeatin 9-riboside O-glucoside

nin in the culture medium, bacteria-free crown-gall cells are capable of sustained proliferation without a supply of these hormones. This autonomy of crown-gall cells with respect to hormones is apparently due to their ability to synthesize endogenous auxin and cytokinin (Nakajima et al. 1979). Further evidence for a role of hormones in tumorous growth comes from studies on tumour morphology. Tobacco crown-gall tissue may grow as an unorganized callus or as an organ-forming teratoma, depending on the strain of initiating bacterium. Amasino and Miller (1982), using bioassay methods, found that tobacco tumour tissues of differing morphology contained characteristic levels of auxin and cytokinin, which could be related to the effects of exogenous hormones on the morphology of cultured nontumorous tissue. Moreover, certain loci in the T-DNA have recently been discovered which control tumour morphology in tobacco. Akiyoshi et al. (1983), using radioimmunoassay in conjunction with high-performance liquid chromatography (HPLC), obtained analytical data which indicate that these T-DNA loci act by controlling tumour auxin and cytokinin levels. The identity of the cytokinins in tobacco crown-gall tissue has been the subject of several investigations. Zeatin riboside (ZR) was the major cytokinin identified by mass-spectrometric analysis of tissue components active in cytokinin bioassays (Nakajima etal. 1979; Einset 1980; Scott etal. 1980a). However, the analytical procedures employed in these studies suffer from two major defects. Firstly, they would not permit detection of cytokinin metabolites which cause no response in the bioassay. Secondly, methods for the rigorous analysis of the cytokinin nucleotide fraction were not available. Studies on the metabolism of exogenous radioactively labelled cytokinins in nontumorous tobacco cultures have revealed the nucleotides and

346

the biologically inactive 7-glucosides as the most abundant metabolites (Fox etal. 1973; Laloue et al. 1974, 1975, 1977, 1981; Laloue and Pethe 1982). Thus, the principal metabolites of exogenous cytokinins in tobacco cells correspond to those metabolic species neglected in the analyses of endogenous tobacco crown-gall cytokinins. In analyses of the cytokinins of Vinca rosea (Catharanthus roseus L.) crown gall tissue (Scott et al. 1980b, 1982a, b), we have employed HPLC and mass spectrometric techniques which have revealed the presence of appreciable quantities of zeatin riboside 5"-monophosphate (ZMP) and of the biologically inactive 9-glucoside of zeatin (Z9G) in addition to the active, basic cytokinins initially reported in this tissue by Peterson and Miller (1977). Precise quantification of these cytokinins was achieved by the use of isotopically labelled internal standards which could be distinguished from the natural compounds in the massspectrometric analysis. In the present paper we describe the application of these techniques to the analysis of the endogenous cytokinins of tobacco crown-gall tissue. Our findings confirm that ZMP and zeatin 7-glucoside (Z7G) are present in this tissue in greater quantities than the previously identified ZR. Materials and methods Synthesis of labelled cytokinins. The synthesis of [2H2]ZMP involved preparation of the acetate salt of 4-amino-4,4-[2H2]-2methylbut-2-enol by the procedure of Summons et al. (1979a) followed by condensation of this compound with 6-chloropurine riboside 5'-monophosphate as described by Scott and Horgan (1981). This procedure yielded a small proportion ( < 10%) of the cis isomer of [2H2]ZMP, which was removed using reversed-phase HPLC. In the synthesis of [aH2]dihydrozeatin 9riboside 5" monophosphate (DHZMP), the above [2H2]aminoalcohol was reduced in the free amine form by stirring a methanolic solution for 3 d under hydrogen with a catalyst of palladium on carbon. The proportion of unlabelled compound yielded by these syntheses was determined by mass spectrometry to be less than 1%. 15N4_labelled zeatin, ZR, zeatin O-glucoside (ZOG), zeatin 9-riboside O-glucoside (ZROG) and Z9G were synthesized as described previously (Scott and Horgan 1980; Scott etal. 1982a). [lSN4]Z7G was prepared biosynthetically. Two l l - d old seedlings of Raphanus sativus were allowed to take up 0.87 mg of [lSN4]zeatin in 0.5 cm 3 H20, and extracted after 5 d with 80% methanol. [15N4]Z7G (0.17 mg) and [15N4]ZOG (0.37 mg) were isolated by reversed- and normal-phase HPLC. In the synthesis of 1SN4_labelled DHZ, 4-amino-2-methylbut-2enol was hydrogenated as described above prior to condensation with [15N4]6.chloropurine (Scott and Horgan 1980). 1SN~labelled dihydrozeatin 9-riboside (DHZR) and dihydrozeatin 7-glucoside (DHZ7G) were prepared by the procedures used for the analogous labelled zeatin glycosides. Resolution of the enantiomers of [15N4]DHZ7G occurred during the reversedphase HPLC purification of this compound. Two peaks (at

I.M. Scott and R. Horgan: Crown-gall cytokinins Table 1. Mass spectra of synthetic isotopically labelled cytoki-

nins, obtained by GC-MS of the derivative indicated. The m/e values of the principal ions only are listed, diagnostic ions of weaker intensity being omitted. Relative intensities are given in brackets. TMSi = trimethylsilyl Permethyl-[2H2]ZR: 423 (M.+, 6.8), 393 (16), 392 (71), 349 (3.3), 248 (4.4), 219 (15), 218 (100), 204 (7.5), 175 (8.8), 150 (5.2), 148 (6.0), 101 (11), 71 (9.2), 69 (8A), 45 (22). Permethyl-[2H2]DHZR: 425 (M +, 17), 410 (24), 394 (33), 338 (24), 280 (33), 252 (50), 236 (41), 220 (El), 178 (37), 174 (SO), 164 (100), 143 (23), 114 (32), 101 (28), 71 (23), 45 (59). Permethyl-[15N4]DHZ: 267 (M +, 11), 252 (15), 236 (18), 194 (30), 181 (20), 180 (100), 167 (14), 152 (8.9), 139 (8.8), 138 (8.4), 137 (10). Permethyl-[15N4]DHZR: 427 (M +, 16), 412 (22), 396 (32), 340 (24), 282 (34), 254 (46), 238 (39), 222 (21), 180 (58), 174 (19), 166 (i00), 143 (27), 114 (29), 101 (25), 45 (43). Penta-TMSi-[15N4]Z7G: 745 (M +, 4.9), 730 (1.5), 656 (5.2), 642 (4.5), 614 (2.5), 450 (0.6), 410 (0.7), 217 (9.9), 147 (13), 75 (20), 73 (100). Penta-TMSi-[15N,]DHZ7G: 747 (M +, 3.0), 732 (2.1), 658 (1.7), 657 (1.2), 603 (3A), 450 (1.4), 440 (2.9), 370 (2.9), 361 (3A), 296 (5.0), 217 (21), 147 (21), 75 (22), 73 (100).

12.4 and 13.3 min), each with UV and mass spectra characteristic of [15N4]DHZ7G ' were obtained using a reversed-phase system involving a 20-min gradient of 5-8% acetonitrile. The earlier of these two peaks had the same retention time as the isolated natural enantiomer of DHZ7G. Previously unreported mass spectra of synthetic labelled cytokinins are given in Table 1. Other mass spectra are given in Scott et al. (1982a).

Plant material. Cultured crown-gall tissue of Nicotiana tabacum L. cv. Wisconsin 38 (initiated with Agrobacterium tumefaciens strain B6) was obtained from Dr. A. Goldmann, C.N.R.A., Versailles, France. The culture was maintained on solidified medium of Murashige and Skoog (1962) under diffused light at 25 ~ C. Samples of tissue were taken for analysis three weeks after subculture.

Purification procedure. For the qualitative analyses, fresh tissue was homogenized in methanol and basic cytokinins were purified from the extract using cation exchange on cellulose phosphate, butan-l-ol partitioning and Sephadex LH-20 column chromatography as described in detail by Scott et al. (1980b). Fractions (30 crn 3) were collected from the Sephadex LH-20 column and 1 cm 3 of each was bioassayed using the Amaranthus test of Biddington and Thomas (1973). Active fractions were further analysed by reversed-phase HPLC as described below. For the quantitative analyses, the tissue was frozen in liquid nitrogen and extracted using Bieleski's solvents (Scott et al. 1982 a). Internal standards were added at the start of the extraction. Basic cytokinins were isolated and analysed as described by Scott et al. (1982a). The acidic-neutral fraction from the cellulose-phosphate step was chromatographed on a column of Dowex l-X8 (50-100 mesh, formate form, 36 cm long, 2.5 cm diameter) eluted with a linear gradient of 10 mM HCOOH (550 cm 3) to 8 M HCOOH (450 cm3). The 360 to 456 cm 3 fraction of the eluate was reduced to dryness in vacuo at 37~ C. Cytokinin riboside 5'-monophosphates were purified from this fraction by reversed-phase HPLC (see below), and then enzy-

I.M. Scott and R. Horgan: Crown-gall cytokinins

347

Table 2. Mass-spectral ions monitored in quantitative analyses Compound

Permethyl-zeatiu Permethyl-ZR Permethyl-ZOG Permethyl-Z9G Penta-TMSia-Z7G Permethyl-DHZ Permethyl-DHZR Penta-TMSi-DHZ7G

Origin of ion

M.+ M.+ M.+ M.+ M.+ M.+ M.+ M.+

Ion m/e value

--

.OCH

3

--

.OCH

3

------

-O-glucosyl-(CH3) 4 "OCH 3 -CH2OTMSi -OCH 3 "OCH 3 "CHzOTMSi

Natural compound

tSN4-compound

230 390 230 434 638 232 392 640

234 394 234 438 642 236 396 644

2H2-compound

392

394

a TMSi = trimethylsilyl matically hydrolysed to the riboside forms by incubation with 0.2 units of calf intestinal alkaline phosphatase (Sigma Chemical Co., Poole, Dorset, U K ) in 0.5 cm 3 of 0.1 M ethanolamineHC1 buffer (pH 10.4) at 37 ~ C for 2 h. The resultant ribosides were then analysed by the H P L C and coupled gas chromatography-mass spectrometry (GC-MS) procedures used for the equivalent compounds in the basic fraction. For the analysis of zeatin riboside 5' mono-, di- and triphosphates, tissue frozen in liquid nitrogen was extracted using the modified Bieleski solvent of Laloue and Pethe (1982). The acidic-neutral fraction of the extract was resolved into mono-, di- and triphosphate nucleotides using diethylaminoethyl (DEAE) cellulose chromatography as described by Palni et al. (1982a). The nucleotides in each fraction were converted to the riboside form using alkaline phosphatase. After addition of [~SN4]ZR as an internal standard, the nucleotide-derived Z R in each fraction was analysed by HPLC and GC-MS.

High-performance liquid chromatography was carried out using two LDC Constametric III pumps controlled by a Gradient Master, coupled to a Spectromonitor III U V detector operating at 265 nm (Laboratory Data Control, Stone, Staffs., UK). Reversed-phase H P L C was carried out using a column (150 m m long, 10 m m diameter) of Apex ODS (Jones Chromatography, Llanbradach, Glamorgan, U K ) eluted at a solvent flow-rate of 5 cm 3 m i n - a. The standard solvent system for basic cytokinins involved a linear 20-min gradient of 7-11% acetonitrile in H 2 0 (adjusted to pH 7 with triethylammonium hydrogen carbonate). Minor modifications were made to this standard solvent programme according to the range of compounds analysed (e.g. see Fig. 1 b). Cytokinin riboside 5'-monophosphates were purified using a linear 20-rain gradient of 0-20 % methanol in 0.1 M acetic acid. Retention times were: Z M P = l l . 2 min; D H Z M P = 12.4 rain. Further details on these chromatographic systems may be found in Horgan and Kramers (1979) and Scott and Horgan (1981). Normal-phase H P L C of basic cytokinins on Partisil 10 PAC (Whatman, Maidstone, Kent, U K ) has been described previously (Horgan and Kramers 1979; Scott et al. 1980b). Normal-phase H P L C of permethylated cytokinins was carried out using a column (150 m m long, 4.5 m m diameter) of Spherisorb 5 g (PhaseSep, Queensferry, Clwyd, U K ) eluted at 2 cm 3 m i n - 1 with hexane: propan-2-ol: triethylamine (Martin et al. 1981), using a solvent composition of 95:5:0.1 (by vol.) for all compounds except the permethyl bases, for which a composition of 85:15:0.1 was used.

Chemical treatments. Isolated Z7G was hydrolysed using cation-exchange resin and glucose was detected by glucose oxidase

following thin-layer chromatography of the hydrolysate, using the procedure of Letham et al. (1975). Chemical degradation of isolated Z M P to the base using sodium periodate was carried out as described by Palni et al. (1983a). Permethyl and trimethylsilyl derivatives of cytokinins were prepared as described previously (Scott et al. 1980b, 1982a).

Spectrometry. Ultraviolet spectra were obtained using a Beckman DU-8 spectrophotometer (Beckman-Riic, High Wycombe, Bucks., UK). Gas-chromatography-mass spectrometry was carried out on a Kratos MS25 mass spectrometer (Kratos, Manchester, U K ) coupled to a gas chromatograph (model 4200; Carlo Erba, Milan, Italy) via a single-stage glass jet separator maintained at 230 ~ C. Mass spectrometry conditions were: resolution 600; ionizing voltage 70 eV; source temperature 200 ~ C. Gas-chromatography systems were developed using both packed columns of OV-1 material (Scott et al. 1982a) and SGE bonded phase capillary columns (0.5 gm film, 3 m m internal diameter, 0.5 kg cm -2 inlet pressure of helium carrier gas) with OCL1 or G r o b injectors (SGE, Milton Keynes, UK). Retention times on capillary G C were as follows. On a 7-m BPI column at 300 ~ C, trimethylsilyl derivatives of D H Z 7 G and Z 7 G eluted after 3.12 and 3.58 min, respectively. On a 10-m BP5 column programmed at 8 ~ C min-~ from 160 ~ C, permethyl derivatives of dihydrozeatin (DHZ) and zeatin eluted after 4.43 and 5.23 min; on the same column programmed at 8 ~ C min 1 from 215 ~ C, permethyl derivatives of D H Z R and Z R eluted after 5.01 and 5.67 min. Table 2 lists the ions used for selected-ion monitoring. Calibration curves for the isotope-dilution procedures were constructed by the method of Pickup and McPherson (1977).

Results

Qualitative analysis. Column chromatography of the basic compounds extracted from tobacco crown-gall tissue revealed a major peak of cytokinin activity with the same elution volume as ZR, while lesser peaks were associated with the elution volumes of zeatin and its glucosides (Fig. i a). The active fractions were further analysed by reversed phase HPLC. The fractions with the elution volumes of zeatin and its riboside yielded peaks of activity with similar HPLC retention times to these

348

I.M. Scott and R. Horgan: Crown-gall cytokinins

ZOG ZR

0.1

Z

t

--01 ~JgBAP

Table 3. Mass spectra of natural cytokinins isolated from tobacco crown gall tissue, obtained by GC-MS of the derivative indicated. The m/e values of the principal ions only are listed, diagnostic ions of weaker intensity being omitted. Relative intensities are given in brackets. TMSi = trimethylsilyl Permethyl-zeatin: 261 (M.+, 7.8) 231 (15), 230 (100), 188 (22), 164 (12), 162 (8.0), 134 (7.5), 107 (9.6), 95 (13), 81 (8.4), 67 (8.4). Permethyl-ZR: 421 (M.+, 6.7), 391 (17), 390 (75), 348 (6.5), 246 (5.3), 217 (14), 216 (100), 202 (7.7), 174 (16), 148 (5.4), 101 (8.0), 71 (6.1), 67 (5.8), 45 (38).

Z

0

300

(J E o.09 b

zo.__G

o

Permethyl-ZOG: 465 (M +, 1.2), 434 (1.9), 231 (15), 230 (100), 188 (8.9), 101 (7.6), 71 (11), 45 (17).

600 ELUTIONVOLUMEcm3 z~6

--

0.1~9BAP

Penta-YMSi-Z7G : 741 (M.+, 4.6), 726 (1.2), 652 (5.3), 638 (3.7), 610 (2.0), 450 (2.5), 406 (1.9), 217 (37), 147 (35), 75 (80), 73 (100). Permethyl-DHZ: 263 (M.+, 10), 248 (13), 232 (17), 190 (31), 177 (22), 176 (100), 163 (15), 148 (9.0), 135 (10), 134 (8.3), 133 (9.3).

LL

-0

0

tO

MINUTES

2'0

Fig. 1. a Amaranthus cytokinin bioassay of basic compounds from tobacco crown-gall tissue after Sephadex LH-20 chromatography. Bars indicate elution volumes of authentic standards. Responses yielded by benzylaminopurine (BAP) controls are indicated at right-hand side. b Amaranthus cytokinin bioassay of glucoside fraction (270-420 cm 3) from Sephadex LH-20 chromatography (a) after preparative reversed-phase HPLC on Apex ODS eluted with HzO (pH 7) containing a linearly increasing concentration of acetonitrile (5-9% over 20 min). Bars indicate retention times of authentic standards. Responses yielded by BAP controls are indicated at right-hand side. Z, zeatin ;ZR, zeatin 9-riboside; ZOG, zeatin O-glucoside; ZROG, zeatin 9-riboside O-glucoside

compounds, and their identities were confirmed by GC-MS as the permethyl derivatives (Table 3). Figure i b shows the HPLC analysis of the glucoside fraction. The cytokinin with the retention time of Z O G was permethylated and identified by GCMS (Table 3). The active fraction with the retention time of Z R O G was derivatized and permethyl Z R O G was purified by normal-phase HPLC prior to mass spectrometry by the direct-probe technique. Although a full characteristic mass spectrum was obtained, this procedure did not achieve sufficient purification to remove background contamination. However, the occurrence of this cytokinin in the tissue was confirmed by GC-MS of Z R released following fl-glucosidase treatment of Z R O G isolated in the isotope-dilution analyses. The other peak of activity seen in Fig. 1 b repre-

Permethyl-DHZR: 423 (M.+, 22), 408 (24), 392 (35), 336 (29), 278 (40), 250 (49), 234 (44), 176 (62), 174 (26), 162 (100), 143 (28), 114 (34), 101 (26). Penta-TMSi-DHZ7G: 743 (M.+, 7.7), 728 (6.3), 654 (3.9), 653 (3.4), 599 (6.0), 450 (4.7), 436 (5.1), 366 (4.6), 361 (5.4), 292 (5.5), 217 (38), 147 (36), 75 (41), 73 (100). Permethyl-AZ-isopentenyladenosine: 392 (24), 391 (M.+, 100), 376 (11), 348 (19), 246 (11), 218 (27), 217 (76), 216 (73),

202 (75), 175 (26), 174 (81), 148 (26), 101 (24), 98 (20), 45 (44).

sents a very active unknown cytokinin, which occurs in such minute quantities that we have so far been unable to obtain an identification. The possibility of cytokinin 3-glucoside formation in tobacco cells has been discussed by Letham et al. (1975), and the unknown compound has the HPLC characteristics and fl-glucosidase susceptibility of the 3-glucoside of zeatin. However, on the present evidence this suggestion remains speculative. The chromatogram of UV-absorbing material obtained for the same HPLC analysis of the glucoside fraction contained a prominent peak at the retention time of the 7-glucoside of zeatin, which is inactive in the bioassay. Gas chromatographymass spectrometry of the trimethylsilyl derivative of this compound yielded a mass spectrum indicative of Z7G (Table 3). The compound exhibited a UV absorption spectrum characteristic of an N6,7-substituted adenine, 2m,~ occurring at 274 nm in 75% ethanol, and at 281 nm in 0.1 M acetic acid (Laloue et al. 1975). Release of D-glucose following acid hydrolysis of the compound was demonstrated using a glucose-oxidase test. In addition, the compound had the same retention time as Z7G on normal-phase HPLC using Partisil 10 PAC. On

I.M. Scott and R. Horgan: Crown-gall cytokinins

the basis of UV absorbance it was apparent that Z7G occurred in this tissue in much greater quantities than did the active glucosides detected by bioassay. A minor peak of UV-absorbing material occurred in the HPLC chromatogram of the glucoside fraction at the retention time of Z9G. However, this compound had a UV absorption spectrum characteristic of an N6,7-substituted adenine (see above). Analysis by GC-MS of the trimethylsilyl derivative of this compound yielded a mass spectrum readily attributable to the 7-glucoside of dihydrozeatin (DHZ7G), shown in Table 3. This is the first reported identification of DHZ7G in plant tissue. A further extraction was therefore undertaken to investigate the presence of DHZ metabolites as minor cytokinin components to tobacco crowngall tissue. The appropriate fractions from Sephadex LH-20 chromatography were analysed by reversed-phase HPLC, fractions with the retention times of DHZ metabolites being collected. Because of the small quantities of these compounds in the extract, their purity was increased prior to GC-MS analysis by normal-phase HPLC of their permethyl derivatives. Dihydrozeatin and its riboside were identified by this procedure (Table 3), though no evidence was obtained for the O-glucoside metabolites of these compounds. A similar procedure was adopted to investigate the occurrence of A2-isopentenyladenine and A2isopentenyladenosine in the tissue. A small quantity of AZ-isopentenyladenosine was isolated and identified (Table 3), though no A2-isopentenyladenine was found. The identification of cytokinin nucleotides in tobacco crown-gall tissue is based on the isotopedilution analyses described in the next section. Quantitative analysis. Substantial and variable losses of cytokinins occur during their purification from plant extracts, and this problem is best overcome by an internal-standard system. We have described the use of 15N4-1abelled analogues in the mass-spectrometric quantification of zeatin glycosides (Scott and Horgan 1980; Scott et al. 1982a). Additional problems arise in the analysis of cytokinin nucleotides. The lack of effective techniques for the isolation of the small quantities of cytokinin nucleotides present in the acidic-neutral fraction of plant extracts has resulted in a general neglect of the analysis of these compounds. We have therefore developed HPLC methods for the purification of cytokinin riboside 5'-monophosphates (Scott and Horgan 1981). A further problem arises from

349

R-CH2,

/C'-CH-CH2 CH3 .X..~.H2~ I

R' Fig. 2. Sites of isotopic labelling in internal standards. The endocyclic purine N-atoms (marked with asterisks) are labelled in the 15N4.cytokinins" The ZH2-cytokinins carry two 2H-atoms at the site on the side chain marked with an asterisk. R and R' are substituents characteristic of the particular cytokinin

the enzymatic hydrolysis of nucleotides to ribosides which may occur in plant extracts (Horgan 1978; Laloue and Pethe 1982). We have chosen to use 2H2-1abelled internal standards for the massspectrometric quantification of cytokinin riboside 5'-monophosphates, so that nucleotide hydrolysis in the extract would be detectable by the appearance of ZH2-1abelled cytokinin ribosides. Our use of these procedures in the analysis of V. rosea crown-gall cytokinins was the first reported quantification of endogenous cytokinin nucleotides (Scott et al. 1982b). The sites of isotopic labelling in the internal standards used in this study are shown in Fig. 2. The endocyclic purine nitrogens are labelled in the 15N4-analogues (Scott and Horgan 1980), while the 2H2-analogues are labelled in the isopentenyl side chain (Summons et al. 1979a). Because only two atoms are labelled in the 2H2-internal standards, slight curvature was seen in calibration curves. The tobacco crown-gall tissue was first analysed for zeatin metabolites. The tissue was extracted using Bieleski's solvents to inactivate phosphatases (Horgan 1978), measured quantities of internal standards being added at the commencement of extraction. Internal standards were added for all the zeatin metabolites detected in the qualitative analysis, together with ZMP and ZgG. The basic and acidicneutral fractions were separated using cellulose phosphate. Following further purification by solvent partitioning and column chromatography, the basic cytokinins were individually isolated using reversed-phase HPLC (Fig. 3). Each cytokinin sample was derivatized and the ratio of natural compound to labelled internal standard was determined by selected-ion monitoring. The results are given in Table 4. No isotopic dilution of the [15N4]ZgG standard with natural compound was

350

I.M. Scott and R. Horgan: Crown-gall cytokinins

I

100

ZTG

O

IX5 390

ZR

392

421 423

o

lO

2o

MINUTES

Fig. 3. Separation of zeatin glycosides in the purified crown-gall extract by preparative reversed-phase HPLC on Apex ODS eluted with HzO (pH 7) containing a linearly increasing concentration of acetonitrile (7-11% over 20 min). The UV absorbance of the eluate was monitored at 265 nm. Z7G, zeatin 7-glucoside; ZOG, zeatin O-glucoside; Z, zeatin; ZROG, zeatin 9-riboside O-glucoside; ZR, zeatin 9-riboside

>,. I.-U3 Z LU I-,Z

I 390

I

430

IJJ I-" .< .,-I LU rr"

~oo

b

IX5 390 394

Table 4. Cytokinin levels in Nicotiana tabacum crown-gall tissue, as estimated by mass spectrometry Cytokinin

,

410

421 425

Quantity per g flesh weight of tissue (nmol)

Zeatin metabolites ZMP Z7G ZR Zeatin ZROG ZOG

0.807 0.203 0.130 0.034 0.024 0.016

Dihydrozeatin metabolites DHZMP DHZ7G DHZR DHZ

0.037 0.028 0.022 0.007

observed, the limit of detection in this analysis being 1.8 pmol per g tissue. Zeatin 9-riboside 5"-monophosphate was purified from the acidic-neutral fraction by Dowex 1-X8 column chromatography, followed by reversed-phase HPLC. After treatment with alkaline phosphatase, the compound was re-isolated as ZR by reversed-phase HPLC and then analysed by the mass-spectrometric procedure used for the basic compounds. The identity and purity of the compounds quantified by selected-ion monitoring was confirmed by scanning GC-MS analyses. Figure 4 shows details of the mass spectra obtained in the analyses of the ZMP and ZR samples (the former

I11, 390

Ill]. 430

410

role Fig. 4a, b. Peak clusters in mass spectra obtained by GC-MS of permethyl Z R samples. Internal standards of [ZHz]ZMP and [15N4]ZR were added to the same tobacco crown-gall tissue extract, and these compounds were then purified, and analysed by GC-MS, as described in Materials and methods. The ZMP sample was analysed as Z R following enzymatic hydrolysis. a M.+ and [M.+ - .OCH3] peak clusters in the mass spectrum obtained for the hydrolysed, permethylated ZMP sample, b M.+ and [M.+ -- .OCH3] peak clusters in the mass spectrum obtained for the permethylated Z R sample. The peaks at m/e 421 and 390 are derived from the natural compound, while those occurring 2 or 4 m/e units higher are derived from the ZH2 or 15N4 internal standards, respectively. The peaks at m/e 423 and 392 in b do not differ in relative intensity from those in the mass spectrum of unlabelled permethyl-ZR

being analysed following phosphatase treatment). Since no contribution from [2Hz]ZR is detectable in the mass spectrum of the ZR sample, it may be concluded that no hydrolysis of ZMP occurred in the extraction. It was generally found that nucleotide breakdown could be prevented providing tissue was frozen in liquid nitrogen prior to extraction with Bieleski's solvent.

I.M. Scott and R. Horgan: Crown-gallcytokinins In order to confirm that the phosphate group of the extracted Z M P was attached to the 5' position of the ribose moiety of the molecule, a portion of the Z M P sample was chemically degraded to zeatin by periodate oxidation, a reaction which requires the presence of two adjacent hydroxyl groups (Yu and Zamecnik 1960). The ratio of internal standard to natural compound in the reaction product was within 1% of the ratio measured for the riboside obtained from phosphatase treatment of the same Z M P sample. This result excludes the possibility that the phosphate group was attached to the 2' or 3' hydroxyls of the ribose moiety, or to the hydroxyl group of the isopentenyl side chain. The formation of nucleoside 5' di- and triphosphate metabolites of exogenous cytokinins has been demonstrated in tobacco cells (Laloue et al. 1974; Laloue and Pethe 1982). In the absence of labelled standards, we were unable to carry out an unambiguous analysis of endogenous zeatin 9riboside 5' diphosphate (ZDP) and triphosphate (ZTP) in the tobacco crown-gall tissue, but a tentative investigation was made by fractionating a tissue extract into mono-, di- and triphosphate nucleotides using diethylaminoethyl cellulose chromatography (Palni et al. 1983 a). These fractions were hydrolysed to ribosides using phosphatase, and [15Ng]ZR was added to each as an internal standard prior to HPLC and GC-MS. The estimated molar ratio of ZMP: ZDP: ZTP thus obtained was 122:35 : 10. The D H Z metabolites identified in the tissue were quantified using similar procedures to those used for the zeatin metabolites, with 1SNg_labelle d DHZ, D H Z R and D H Z 7 G being used as internal standards. D H Z M P was quantified simultaneously using 2H2-1abelled standard. In the case of these minor cytokinins, it was again found desirable to use HPLC to purify the permethylated compounds prior to GC-MS. The analytical results are given in Table 4. Attempts to detect endogenous A 2-isopentenyladenine metabolites by similar isotope dilution methods were unsuccessful (details not presented), except in the case of A2-isopentenyladenosine, for which an estimate of 5.5 pmol g - ~ was obtained. Discussion

The principal endogenous cytokinins of tobacco crown-gall tissue are metabolites of zeatin, as demonstrated by earlier studies (Nakajima et al. 1979; Einset 1980; Scott et al. 1980a). However, the analytical techniques which we have recently devel-

351 oped revealed that previously neglected types of metabolite were quantitatively more important than the basic, biologically active cytokinins identified in these earlier studies. In fact, the cytokinin content of tobacco crown-gall tissue proved to be comparable to the spectrum of metabolites which arises when cytokinin-requiring tobacco cultures are supplied with radioactively labelled cytokinin (Laloue and Pethe 1982). The major endogenous cytokinin quantified in tobacco crown-gall tissue was ZMP, which was six times more abundant in molar terms than ZR, the compound previously thought to be the major cytokinin in this tissue. In turn, zeatin was present in about one-quarter the quantity of its riboside. We have confirmed these findings in four analyses of this tissue. The high level of endogenous Z M P in this tissue is in accordance with the metabolic experiments of Laloue and co-workers on tobacco cells (Laloue et al. 1977, 1981; Laloue and Pethe 1982). This group has also demonstrated the formation of di- and triphosphate cytokinin nucleotides in tobacco cells (Laloue et al. 1974; Laloue and Pethe 1982). We obtained tentative evidence for the presence of endogenous zeatin nucleotides of this type in the crown-gall tissue, but in smaller quantities than the monophosphate. However, further analytical techniques need to be developed before unambiguous analysis of these compounds can be undertaken. A further correspondence between the cytokinin content of the crown-gall tissue and the metabolism of exogenous cytokinins in tobacco cells is the prominence of 7-glucoside metabolites. Formation of the 7-glucoside of benzyladenine in tobacco callus was first demonstrated by Fox et al. (1973), and the role of this cytokinin metabolite in tobacco cells has been studied by Laloue and co-workers (Gawer et al. 1977; Laloue et al. 1975, 1977, 1981 ; Laloue and Pethe 1982). In the tobacco crown-gall tissue, Z7G was the most abundant cytokinin in the basic fraction. Minor quantities of cytokinin O-glucosides (ZOG and ZROG) were also detected; compounds of this type would not be formed from the particular cytokinins used in the metabolic studies of Fox and Laloue. The 9-glucoside metabolite was not detectable in the crowngall tissue, and it has never been reported in metabolic studies. The dynamics of cytokinin metabolism and the biological activity of the various cytokinin metabolites in tobacco cell cultures have been studied by Laloue and co-workers, and in view of the similarities outlined above it would appear that their findings might be relevant to the naturally occurring

352

cytokinins of crown-gall tissue of this species. Laloue and Pethe (1982) found that in an apparent steady-state situation the nucleotide, riboside and base forms of cytokinins undergo continuous interconversion in tobacco cells. They also obtained evidence that the biologically active form of cytokinin is the base, despite the relatively low levels at which it occurs in tobacco cells. On the other hand, the 7-glucoside form has been demonstrated by Laloue (1977) to be biologically inactive. The rate of reutilization of cytokinin 7-glucosides is extremely slow (Gawer et al. 1977) and their accumulation would appear to represent a terminal inactivation pathway (Laloue et al. 1981; Laloue and Pethe 1982). The analysis reported in the present paper permits an assessment of the qualitative validity of the ttPLC-radioimmunoassay protocol employed by Akiyoshi et al. (1983) to obtain their evidence for the role of cytokinins in crown-gall turnout development. Two main points can be made. Firstly, in order to simplify their analyses, these authors treated their tissue extracts with phosphatase. Therefore it seems probable that most of the ZR detected in these analyses was actually derived from nucleotides. Secondly, 7-glucosides appear not to be cross-reactive in the type of cytokinin radioimmunoassay used in these analyses (Weiler and Spanier 1981), and so appreciable quantities of Z7G may have gone undetected. However, as discussed above, Laloue and Pethe (1982) believe that the cytokinin nucleotide and riboside pools are in metabolic equilibrium in tobacco cells, while Z7G simply represents an inactivated form of cytokinin. Considered in these terms, the findings of Akiyoshi et al. (1983) should be biologically meaningful. Similar considerations may be applied to the bioassay procedure of Amasino and Miller (1982), which in fact revealed a predominance of nucleotide-like cytokinin activity in tobacco crown-gall tissue, but would not have detected the inactive 7-glucoside. Minor quantities of D H Z metabolites occurred as endogenous compounds in the tobacco crown gall tissue. Dihydrozeatin metabolites are produced in similarly low quantities by crown-gall tissue of V. rosea (Palni and Horgan 1982) and D a tura innoxia (Palni et al. 1983 b). These compounds are formed by reduction of the zeatin side-chain (Sondheimer and Tzou 1971), but the importance of this conversion remains uncertain. It was also established that very small quantities of AZ-isopentenyladenosine occur in tobacco crown-gall tissue. Again, similar results have been obtained for crown-gall tissue of V. rosea (Scott

I.M. Scott and R. Horgan: Crowrt-gall cytokinins

et al. 1982b) and D. i n n o x i a (Palni et al. 1983b). Recent studies indicate that A2-isopentenyladenine metabolites represent the initial stage of cytokinin biosynthesis (Chen 1982; Palni and Horgan 1983), and Morris et al. (1982) have obtained evidence that the zlZ-isopentenyl transferase responsible for their formation in tobacco crown-gall cells may be coded for by a T-DNA locus. This possibility is supported by the identification of A2-isopentenyladenine metabolites in crown-gall tissues, but the low levels at which they occur imply very rapid hydroxylation to zeatin metabolites. It is interesting that relatively high levels of endogenous AZ-isopentenyladenosine have been demonstrated in a nontumorous, cytokinin-autonomous tobacco culture (Dyson and Hall 1972). The cytokinin content of tobacco crown-gall tissue differs qualitatively from that of V. rosea crown-gall tissue, in which ZR occurs in greater quantities than ZMP, and in which ZOG, ZROG and Z9G occur in the absence of Z7G (Scott et al. 1980b, 1982a, b). A different situation again is found in D. i n n o x i a crown-gall tissue, in which high levels of both Z7G and Z9G occur, together with lesser quantities of the O-glucosides (Palni et al. 1983 b). Since the range of endogenous cytokinins in tobacco crown-gall tissue reflects the metabolic patterns of nontumorous, cytokinin-requiring tobacco cells, it seems possible to conclude that the metabolic conversions of cytokinins in crowngall cells are determined by the host genome of the transformed cells. This diversity of cytokinin metabolites in crown-gall tissues illustrates the need for critical evaluation of adopted procedures before biological conclusions can be drawn from analytical results. For example, we do not consider that the radioimmunoassay procedure of Weiler and Spanier (1981) was sufficiently comprehensive to justify their contention that some crown-gall tissues can proliferate in the absence of endogenous cytokinin. Their analyses would not have detected various important types of cytokinin, such as O-glucosides, 7glucosides and DHZ metabolites (Weiler and Spanier 1981; E.W. Weiler, Ruhr-Universit/it Bochum, FRG, personal communication). In conclusion, we believe that comprehensive and accurate analysis of endogenous cytokinins provides an essential foundation for an understanding of the regulation and expression of cytokinin activity in plant tissues. As the present paper demonstrates, analytical neglect of any particular class of metabolite may yield an incomplete and misleading picture of the cytokinin content of a tissue. Therefore, the introduction of new analyti-

I.M. Scott and R. Horgan: Crown-gall cytokinins

cal procedures is of fundamental importance for the advancement of our knowledge of cytokinins. In recent years, analytical advances have revealed the prominence of glucosylated cytokinins in plant tissues (Summons et al. 1979b; Scott et al. 1982a). The neglect of cytokinin nucleotide analysis, however, persisted until our development of methods for the isolation and quantification of these compounds (Scott and Horgan 1981; Scott etal. 1982b). Recently, Summons et al. (1983) have reported similar analytical procedures for cytokinin nucleotides, including the valuable development of a chemical-ionization mass-spectrometry technique for the intact ZMP molecule. The possible general importance of cytokinin nucleotides in plant tissues is indicated by our demonstration that ZMP is by far the most abundant cytokinin in tobacco crown-gall tissue. We have recently carried out a similar analysis of Z e a m a y s kernels, finding that ZMP occurs in quantities four times greater than the riboside and base (data not shown). It seems likely, therefore, that the future application of the new analytical techniques for cytokinin nucleotides will result in an extensive reevaluation of the existing cytokinin literature. We thank Mr. J.K. Heald for operation of the mass spectrometer, and Mr. P.E. Williams for technical assistance. We are very grateful to Dr. A. Goldmann for supplying the crown-gall culture. This research forms part of a project financed by the Agricultural and Food Research Council.

References

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I.M. Scott and R. Horgan: Crown-gall cytokinins Summons, R.E., Duke, C.C., Eichholzer, J.V., Entsch, B., Letham, D.S., MacLeod, J.K., Parker, C.W. (1979a) Mass spectrometric analysis of cytokinins in plant tissues. II. Quantitation of cytokinins in Z e a m a y s kernels using deuterium labelled standards. Biomed. Mass Spectrom. 6, 407-413 Summons, R.E., Entsch, B., Parker, C.W., Letham, D.S. (1979b) Mass spectrometric analysis of cytokinins in plant tissues. III. Quantitation of the cytokinin glycoside complex of lupin pods by stable isotope dilution. FEBS Lett. 107, 21-25 Summons, R.E., Palni, L.M.S., Letham, D.S. (1983) Determination of intact zeatin nucleotide by direct chemical ionisation mass spectrometry. FEBS Lett. 151, 122-126 Weiler, E.W., Spanier, K. (1981) Phytohormones in the formation of crown gall tumors. Planta 153, 326-337 Willmitzer, L., De Beuckeleer, M., Lemmers, M., Van Montagu, M., Schell, J. (1980) DNA from Ti plasmid present in nucleus and absent from plastids of crown gall cells. Nature (London) 287, 359-361 Yu, C.-T., Zamecnik, P.C. (1960) A hydrolytic procedure for ribonucleosides and its possible application to the sequential degradation of RNA. Biochim. Biophys. Acta 45, 148-154 Received 21 December 1983; accepted 16 February 1984