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The Metabolism of Kinetin and Benzyladenine in Soybean Callus: Determination by Radioimmunoassay and Radioassay
The Metabolism of Kinetin and Benzyladenine in Soybean Callus: Determination by Radioimmunoassay and Radioassay
1) Department of Honicultural Science, University of Natal, Pietermaritzburg, 3200, Republic of South Africa.
2) UN/CSIR Research Unit for Plant Growth and Development, Department of Botany, University of Natal, Pietermaritzburg, 3200, Republic of South Africa. Received December 17, 1985 . Accepted January 8, 1986
Summary Previous investigations indicated the potential for the use of antisera produced against dihydroribosylzeatin for the quantification of kinetin (K) and benzyladenine (BA). Assays were developed for these synthetic cytokinins and their respective ribosides. Assay measuring range for K and BA was 0.12-46.5pmol and 0.22-44.4pmol, respectively. Similar measuring ranges were obtained for both K and BA riboside (0.07 - 28 pmol). The assays were applied to a study of the metabolism of K and BA in soybean callus cultures. Extract purification was achieved with ion exchange chromatography, and the bases and ribosides separated using thin layer chromatography. The results were in good agreement with those obtained using the more conventional methods of Sephadex LH-20 and HPLC, and indicated rapid metabolism of both K and BA during the 48 h experimental period. No metabolites apart from BA riboside were detected by RIA. HPLC however indicated the presence of other metabolites, and these are reponed in a concurrent paper.
Key words: Soybean callus, cytokinin, kinetin, benzyiadenine, radioimmunoassay, HPLC, me· tabolism.
Introduction
In a previous paper (Hofman et al., 1985) RIAs were described for the quantitation of DHZ and DHZR using antibodies raised in rabbits against a DHZR-BSA conjugate. The antiserum produced also showed high cross-reaction with K and BA, suggesting the potential of this antiserum for the quantitation of these synthetic cytokinins. An assay system of this nature was considered potentially useful because of the advantages of RIA (Weiler, 1982) and the common use of K and BA as synthetic cytokinins in tissue culture and metabolic studies on cytokinins. This paper describes the successful development of a sensitive RIA for the quantitation of K, BA and their ribosides. The assays were used to study the rate of utilisation of K and BA in soybean callus cultures, and the production of the ribosides. The RIA Abbreviations: BA = benzyladenine; BAR = ribosylbenzyladenine; BSA = bovine serum albumin; DHZ = dihydrozeatin; DHZR = ribosyldihydrozeatin; HPLC = high performance liquid chromatography; K = kinetin; KR = ribosylkinetin; RIA = radioimmunoassay; TLC = thin layer chromatography; UV = ultraviolet.
J. Plant Physiol. Vol.
124. pp. 289-298 (1986)
290
P.]. HOFMAN, CLARE FORSYTH and].
VAN
STADEN
results were confirmed in experiments where 14C_K and 14C_BA were fed to callus and the various metabolites subjected to HPLC analysis and radioassay.
Materials and Methods Chemicals [8)4C}kinetin (specific activity 925 MBq mmol- I) and [8_14C}BA (specific activity 495 MBq mmol- I) were obtained from the Radiochemical Centre, Amersham. All other chemicals and equipment were as described previously (Hofman et al., 1985).
Extraction and purification for RIA Redistilled ethanol (80 ml) was added to the 20 ml culture medium containing 5 g callus. The callus was macerated together with the medium and then extracted for 24 h at 4 dc. The extract was then filtered and evaporated to dryness in 'Vacuo at 40°C. The residue was redissolved in 20 ml distilled water, the pH adjusted to 2.5, and purified with Dowex using a method modified from Whenham (1983). The extract was shaken for 1 hr with 5 g of Dowex 50W-X8 (50-100 mesh, H+ form). The supernatant was decanted and the Dowex washed twice by shaking for 15 min with 50 ml of 80 % ethanol. The cytokinins were eluted from the Dowex by shaking for 2 x 30 min with 2 x 50 ml 96 % ethanol: 25 % ammonia: water (80: 11 : 9) and the two eluant fractions combined and evaporated to dryness in 'Vacuo at 40°C. The residue was redissolved in 1 ml methanol and 10,J of this subjected to TLC (silica gel; solvent system n-butanol: 25 % ammonia: water, 6: 1 : 2, upper phase). The required Rf zones were removed, eluted in 5 ml methanol and 0.2 ml assayed by RIA. Using these techniques, percent recoveries were approximately 17 % and 34 % for K and BA, respectively.
Extraction and purification for HPLC The extraction and purification procedures for the preparation of the 14C-BA-treated material were as described previously (Hofman et al., 1985). However, where callus was fed 14C_ K, the Dowex purification step was omitted and the plant material and respective culture medium simply homogenised and allowed to extract overnight at 4°C. The extract was then filtered and the supernatant concentrated to dryness in 'Vacuo at 40°C. The residue was redissolved in 35 % ethanol and fractionated using a Sephadex LH-20 column eluted with 35 % ethanol (Armstrong et al., 1969). For HPLC analysis, the required fractions from the initial Sephadex separation were redissolved in 80,J methanol, 8,J of which was injected onto a Varian MCH-5 column (5 JLm, CIS bonded, 150x4mm i.d.; flow rate Iml min-I) fitted to a Varian 5000 liquid chromatograph. The mobile phase was 100 % water over 4 min, to 10 % acetonitrile over 1 min, then to 30 % acetonitrile over 25 min. Absorbance was recorded with a Varian variable wavelength monitor at 265 nm, fitted with an 8,J flow-through cell. One ml fractions of eluant were collected and 4 ml Beckman Ready Solv EP added. The radioactivity was determined using a Beckman LS 3800 liquid scintillation counter. The plant material, culture conditions, and RIA procedure were identical to that used for DHZ and DHZR (Hofman et al., 1985), with the exception that the appropriately diluted tracer (lH-DHZ) and antibody solutions were combined and 0.2 ml added to each test tube.
Results Assay characteristics Assay characteristics were very similar to those described for DHZ and DHZR (Hofman et al., 1985). The typical within-assay coefficient of variation was decreased from lOA % to 7.6 % (n = 10 using standard to produce B/Bo = 50 %) by mix-
J Plant PhysioL Vol. 124. pp. 289-298 (1986)
Metabolism of kinetin and benzyladenine
291
2
o -1
'0 -2 CDIII ~
2
-I
1
8
o -1
-2
0.1
pmol
Fig.l: Typical standard curves obtained for K (A) and BA (B). The bars indicate the variation with duplicate samples. Where no bars are shown the variation was smaller than the symbol. Bo = binding of tracer to the antibody in the absence of standard. B = binding of tracer to the antibody in the presence of standards. Logit (B/Bo) = In [(B/Bo)/(l-[B/BoJ)]. ing the antibody and tracer solutions prior to dispensing into the test tubes. The typical measuring range of the assay for BA and K was 0.22-44.4pmol (0.OS-10ng) and 0.12-46.5 pmol (0.D2S-l0ng) respectively (Fig. 1 A and Fig. 1 B). The assays for BAR and KR were very similar to their respective bases, with typical measuring ranges of 0.07 - 28 pmol (0.025 -10 ng) and 0.07 - 28.8 pmol (0.025 -10 ng), respectively. Purification ofplant material for RIA
Analysis of the ethanolic extract without prior Dowex purification was investigated initially because of the potential degradation of K during Dowex purification. However, the extract dilution curve did not parallel the standard curve, indicating the presence of contaminants not removed by TLC. These were removed with Dowex treatment, as indicated by parallelism between the extract dilution curve and standard curve. The method of Dowex purification used was chosen to reduce the risk of localized temperature and pH extremes generally associated with the use of a strong cation exchange resin column (Dyson and Hall, 1972). The degradation of 14C_K to adenine using this Dowex procedure was examined, and found to be negligible.
J Plant Physiol. Vol. 124. pp. 289-298 (1986)
292
P. J. HOFMAN, CLARE FORSYTH and J. VAN STADEN
7 6
3 2
o
0- - - -0- - - 0- - - - - -0 - - - - - - - - - - - -0
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24 TIME hours
48
Fig. 2: Mass of K (--e--) and KR (- - - - -0- - - - -) detected in 20 ml culture medium containing 5 g soybean callus at various times of incubation after the addition of 14C-K, as determined by RIA. Bars indicate variation between duplicate samples. Where there are no bars, the variation was smaller than the symbol.
Metabolism ofK by soybean callus Analysis by RIA indicated an 80 % reduction in detectable K during the initial 24 h of incubation (Fig. 2). In the following 24 h period, no further decrease occurred. Kinetin riboside was not detected at any time during the 48 h incubation period. The production of potentially cross-reacting 3- and 9-glucosides (Weiler and Spanier, 1981) was investigated by assaying all Rfs from TLC by RIA. Significant additional assay response was obtained at Rf 0.2-0.25 only (Fig. 3). This corresponded to an intense UV-quenching spot at Rf 0.20-0.23, with consistent intensity throughout the incubation period. This was in contrast to a faint UV-quenching spot at Rf 0.77 0.82 (corresponding to K) which decreased in intensity with longer incubation. Thus the compound at Rf 0.20 - 0.23 was not considered to represent a cross-reacting K metabolite. Analysis by conventional chromatographic techniques (Sephadex LH-20 and HPLC) indicated that 14C_K was rapidly metabolised, and Sephadex LH-20 chromatography revealed that a number of metabolic products appeared as a result (Fig. 4). Each radioactive peak was further analysed by HPLC. No evidence was found to in-
J. Plant Physiol. Vol.
124. pp. 289-298 (1986)
Metabolism of kinetin and benzyladenine
KR
293
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Rf Fig. 3: The immunoreactivity (expressed as K equivalents) of various Rfs from TLC of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 24 h after 14C_K was applied. Bars indicate Rfs at which visible UV-quenching occurred. K = kinetin, KR = ribosylkinetin.
Ado
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~
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6
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0
4
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400
800
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1200
1600
ElUTION VOLUME ml Fig. 4: Sephadex LH-20 separation of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 24 h after 14C-K was applied. The column was eluted with 35 % ethanol. Ade = adenine, Ado = adenosine, K = kinetin.
J Plant Physiol. Vol. 124. pp. 289-298 {1986}
15
5
cr' ,.cr - -.().. - - - - -0- - - - - - - - - - - - -
o
0
48 Fig. 5: Mass of BA (--e--) and BAR (- - - - -0- - - - -) detected in 20 ml culture medium containing 5 g soybean callus at various times of incubation after the addition of 14C-BA, as determined by RIA. Bars indicate variation between duplicate samples. Where there are no bars, the variation was smaller than the symbol.
BAR
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~
~
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I
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Fig. 6: The immunoreactivity (expressed as BA equivalents) of various Rfs from TLC of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 24 h after 14C_BA was applied. Bars indicate Rfs at which visible UV-quenching occurred. BA = benzyladenine, BAR = ribosylbenzyladenine.
1. Plant Physiol. Vol. 124. pp. 289-298 (1986)
Metabolism of kinetin and benzyladenine
295
BA
o
o
400
1200
800
1600
ELUTION VOLUME ml
Fig. 7: Sephadex LH-20 separation of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 12 h after 14C_BA was applied. The column was eluted with 35 % ethanol. BA = benzyladenine, Ade = adenine, Ado = adenosine.
100
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TIME hours Fig. 8: Radioactivity associated with BA (--e--) and with BAR (- - - - -0- - - - -) over the 48 h experimental period as determined by radioassay and HPLC. Values are expressed as a percentage of the total recovered radioactivity.
J. Plant Physiol.
Vol. 124. pp. 289-298 (1986)
P.]. HOFMAN, CLARE FORSYTH and]. VAN STADEN
296
o
® Ade
Ade 12.0
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It) (Q
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o
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Fig. 9: A. HPLC separation of authentic standards (--) and the radioactive peak (- - - - -e- - - --) which, on Sephadex LH-20, had an elution volume of 1240 -1360 ml. See Materials and Methods for the conditions used. B. As for A except that the peak having an elution volume of 960 -1040 ml (Fig. 7) was analysed. Ade = adenine, BA = benzyladenine, BAR = ribosylbenzyladenine.
dicate the presence of KR during the experimental period. The percentage radioactivity associated with K declined from 73.2 % at the start to 9.2 % after 24 h, and then to 0.62 % after 48 h of incubation. Other metabolic products were formed and, although these were further analysed, they are reported on in a concurrent paper (Forsyth and Van Staden, 1986), as the aim of this paper was to compare RIA with more conventional assay procedures. None of these metabolites were of the glucosylated or phosphorylated nature, and the only ribosylated derivative detected was adenosine.
Metabolism of BA by soybean callus Analysis by RIA indicated that BA also underwent a rapid metabolism (Fig. 5). However, in contrast to K, a rapid decrease in detectable BA occurred during the first 12 h, which continued at a reduced rate during the following 36 h. At 48 h a 92 % reduction in BA was observed. BAR was first detected at 6 h, reached a maximum at 12 h, and decreased by 78 % at 48 h. An analysis of the TLC plate revealed the presence of BA and BAR, but, as with K, no other cross-reacting compounds likely to correspond to BA glucosides were detected (Fig. 6). Analysis by conventional chromatography revealed that the rate of metabolism of 14C_BA was similar to that of 14C_K, and after only 12 h incubation a number of metabolites were present (Fig. 7). However, the percentage radioactivity associated with
J. Plant Physiol.
Vol. 124. pp. 289-298 (1986)
Metabolism of kinetin and benzyladenine
297
BA declined during the first 6h of incubation from 82.4% to 25.9%. Thereafter the decline was slower until, at 48 h, only 10.3 % of the tot;li radioactivity was associateJ with BA (Fig. 8 and Fig. 9 A). HPLC analyses revealed that small amounts of BAR were formed and first appeared after 12 h incubation (7.2 % of the total recovered radioactivity). Thereafter the levels declined marginally to 3.5 % after 48 h incubation (Fig. 8 and Fig. 9 B). Again other metabolites were detected and are reported on in a concurrent paper (Forsyth and Van Staden, 1986). No metabolite apart from adenosine responded to acid treatment (Dyson et al., 1972), indicating that none of these products were of a glucosylated, ribosylated or phosphorylated nature. Discussion Neither the antiserum nor the TLC system used had the ability to distinguish between K and DHZ, and their ribosides. However, soybean callus does not inherently contain DHZ or DHZR (Van Staden and Davey, 1977), and thus does not possess the capacity for their synthesis. It is, therefore, highly improbable that soybean callus has the capacity to synthesise DHZ from added K. Therefore the TLC system used was considered adequate for this investigation, despite the fact that K (Rf 0.6) and KR (Rf 0.46) chromatographed very closely with DHZ (Rf 0.66) and DHZR (Rf 0.44). If K analyses are to be conducted on plant tissue likely to contain the dihydro-derivatives, a TLC system which adequately separates these compounds, such as silica gel developed with chloroform: methanol (9 : 2) could be employed. The assays developed proved to be a rapid means of assaying K, BA, and their ribosides. Assay sensitivity was similar to those developed previously (Hofman et al., 1985,1986), despite the fact that the antiserum used was not produced specifically for these synthetic cytokinins. Little sample purification was required. The agreement between the results obtained by RIA and conventional Sephadex LH-20 and HPLC was good. More metabolites were detected when Sephadex LH-20 and HPLC techniques were employed (Forsyth and Van Staden, 1986), not because of the relative sensitivity of the two techniques, but primarily because of the specificity of the antiserum used (Hofman et al., 1985). This is the major drawback of the use of RIA in metabolic studies, and if the detection of metabolites, in addition to the metabolism of the initial compound is required, then additional less specific techniques will be needed. Acknowledgements The financial assistance of the C.S.I.R., Pretoria and the University of Natal Research Fund is gratefully acknowledged.
References ARMSTRONG, D. J., W. J. BURROWS, P. F. EVANS, and F. SKOOG: Isolation of cytokinins from tRNA. Biochem. Biophys. Res. Commun. 37, 451-456 {1969}. DYSON, W. H. and R. H. HAu.: N 6-(L>2-isopentenyl}adenosine: Its occurrence as a free nucleoside in an autonomous strain of tobacco tissue. Plant Physiol. 50, 616-621 {1972}. DYSON, W. H., J. E. Fox, and J. D. MCCHESNEY: Short term metabolism of urea and purine cytokinins. Plant Physiol. 49, 506-513 {1972}.
J. Plant PhysioL VoL 124. pp. 289-298 (1986)
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P. J. HOFMAN, CLARE FoRSYTH and J. VAN STADEN
FORSYTH, C. andJ. VAN STADEN: The metabolism and cell division activity of adenine derivatives • in soybean callus. J. Plant Physiol. 124,275-287 (1986). HOFMAN, P. J., C. FoRSYTH, andJ. VAN STADEN: A radioimmunoassay for dihydrozeatin and dihydrozeatinriboside, and its application to a study of the in vitro metabolism of dihydrozeatin by soybean callus. J. Plant Physiol. 121, 1-12 (1985 a). HOFMAN, P.J., B. C. FEATONBy-SMITII, andJ. VAN STADEN: The development of ELISA and RIA for cytokinin estimation and their application to a study of lunar periodicity in Ecklonia maxima (Osbeck) Papenf. J. Plant Physiol. 122, 455-466 (1986). VAN STADEN, J. and J. E. DAVEY: The metabolism of zeatin and zeatin riboside in soya bean callus. Ann. Bot. 41,1041-1048 (1977). WEILER, E. W.: Plant hormone immunoassay. Physiol. Plant. 54, 230-234 (1982). WEILER, E. W. and K. SPANIER: Phytohormones in the formation of crown gall tumors. Planta 153,326-337 (1981). WHENHAM, R. J.: Evaluation of selective detectors for the rapid and sensitive gas chromatographic assay of cytokinins, and application to the analysis of cytokinins in plant extracts. Planta 157, 554-560 (1983).
J. Plant Physiol. Vol. 124. pp. 289-298 (1986)
1) Department of Honicultural Science, University of Natal, Pietermaritzburg, 3200, Republic of South Africa.
2) UN/CSIR Research Unit for Plant Growth and Development, Department of Botany, University of Natal, Pietermaritzburg, 3200, Republic of South Africa. Received December 17, 1985 . Accepted January 8, 1986
Summary Previous investigations indicated the potential for the use of antisera produced against dihydroribosylzeatin for the quantification of kinetin (K) and benzyladenine (BA). Assays were developed for these synthetic cytokinins and their respective ribosides. Assay measuring range for K and BA was 0.12-46.5pmol and 0.22-44.4pmol, respectively. Similar measuring ranges were obtained for both K and BA riboside (0.07 - 28 pmol). The assays were applied to a study of the metabolism of K and BA in soybean callus cultures. Extract purification was achieved with ion exchange chromatography, and the bases and ribosides separated using thin layer chromatography. The results were in good agreement with those obtained using the more conventional methods of Sephadex LH-20 and HPLC, and indicated rapid metabolism of both K and BA during the 48 h experimental period. No metabolites apart from BA riboside were detected by RIA. HPLC however indicated the presence of other metabolites, and these are reponed in a concurrent paper.
Key words: Soybean callus, cytokinin, kinetin, benzyiadenine, radioimmunoassay, HPLC, me· tabolism.
Introduction
In a previous paper (Hofman et al., 1985) RIAs were described for the quantitation of DHZ and DHZR using antibodies raised in rabbits against a DHZR-BSA conjugate. The antiserum produced also showed high cross-reaction with K and BA, suggesting the potential of this antiserum for the quantitation of these synthetic cytokinins. An assay system of this nature was considered potentially useful because of the advantages of RIA (Weiler, 1982) and the common use of K and BA as synthetic cytokinins in tissue culture and metabolic studies on cytokinins. This paper describes the successful development of a sensitive RIA for the quantitation of K, BA and their ribosides. The assays were used to study the rate of utilisation of K and BA in soybean callus cultures, and the production of the ribosides. The RIA Abbreviations: BA = benzyladenine; BAR = ribosylbenzyladenine; BSA = bovine serum albumin; DHZ = dihydrozeatin; DHZR = ribosyldihydrozeatin; HPLC = high performance liquid chromatography; K = kinetin; KR = ribosylkinetin; RIA = radioimmunoassay; TLC = thin layer chromatography; UV = ultraviolet.
J. Plant Physiol. Vol.
124. pp. 289-298 (1986)
290
P.]. HOFMAN, CLARE FORSYTH and].
VAN
STADEN
results were confirmed in experiments where 14C_K and 14C_BA were fed to callus and the various metabolites subjected to HPLC analysis and radioassay.
Materials and Methods Chemicals [8)4C}kinetin (specific activity 925 MBq mmol- I) and [8_14C}BA (specific activity 495 MBq mmol- I) were obtained from the Radiochemical Centre, Amersham. All other chemicals and equipment were as described previously (Hofman et al., 1985).
Extraction and purification for RIA Redistilled ethanol (80 ml) was added to the 20 ml culture medium containing 5 g callus. The callus was macerated together with the medium and then extracted for 24 h at 4 dc. The extract was then filtered and evaporated to dryness in 'Vacuo at 40°C. The residue was redissolved in 20 ml distilled water, the pH adjusted to 2.5, and purified with Dowex using a method modified from Whenham (1983). The extract was shaken for 1 hr with 5 g of Dowex 50W-X8 (50-100 mesh, H+ form). The supernatant was decanted and the Dowex washed twice by shaking for 15 min with 50 ml of 80 % ethanol. The cytokinins were eluted from the Dowex by shaking for 2 x 30 min with 2 x 50 ml 96 % ethanol: 25 % ammonia: water (80: 11 : 9) and the two eluant fractions combined and evaporated to dryness in 'Vacuo at 40°C. The residue was redissolved in 1 ml methanol and 10,J of this subjected to TLC (silica gel; solvent system n-butanol: 25 % ammonia: water, 6: 1 : 2, upper phase). The required Rf zones were removed, eluted in 5 ml methanol and 0.2 ml assayed by RIA. Using these techniques, percent recoveries were approximately 17 % and 34 % for K and BA, respectively.
Extraction and purification for HPLC The extraction and purification procedures for the preparation of the 14C-BA-treated material were as described previously (Hofman et al., 1985). However, where callus was fed 14C_ K, the Dowex purification step was omitted and the plant material and respective culture medium simply homogenised and allowed to extract overnight at 4°C. The extract was then filtered and the supernatant concentrated to dryness in 'Vacuo at 40°C. The residue was redissolved in 35 % ethanol and fractionated using a Sephadex LH-20 column eluted with 35 % ethanol (Armstrong et al., 1969). For HPLC analysis, the required fractions from the initial Sephadex separation were redissolved in 80,J methanol, 8,J of which was injected onto a Varian MCH-5 column (5 JLm, CIS bonded, 150x4mm i.d.; flow rate Iml min-I) fitted to a Varian 5000 liquid chromatograph. The mobile phase was 100 % water over 4 min, to 10 % acetonitrile over 1 min, then to 30 % acetonitrile over 25 min. Absorbance was recorded with a Varian variable wavelength monitor at 265 nm, fitted with an 8,J flow-through cell. One ml fractions of eluant were collected and 4 ml Beckman Ready Solv EP added. The radioactivity was determined using a Beckman LS 3800 liquid scintillation counter. The plant material, culture conditions, and RIA procedure were identical to that used for DHZ and DHZR (Hofman et al., 1985), with the exception that the appropriately diluted tracer (lH-DHZ) and antibody solutions were combined and 0.2 ml added to each test tube.
Results Assay characteristics Assay characteristics were very similar to those described for DHZ and DHZR (Hofman et al., 1985). The typical within-assay coefficient of variation was decreased from lOA % to 7.6 % (n = 10 using standard to produce B/Bo = 50 %) by mix-
J Plant PhysioL Vol. 124. pp. 289-298 (1986)
Metabolism of kinetin and benzyladenine
291
2
o -1
'0 -2 CDIII ~
2
-I
1
8
o -1
-2
0.1
pmol
Fig.l: Typical standard curves obtained for K (A) and BA (B). The bars indicate the variation with duplicate samples. Where no bars are shown the variation was smaller than the symbol. Bo = binding of tracer to the antibody in the absence of standard. B = binding of tracer to the antibody in the presence of standards. Logit (B/Bo) = In [(B/Bo)/(l-[B/BoJ)]. ing the antibody and tracer solutions prior to dispensing into the test tubes. The typical measuring range of the assay for BA and K was 0.22-44.4pmol (0.OS-10ng) and 0.12-46.5 pmol (0.D2S-l0ng) respectively (Fig. 1 A and Fig. 1 B). The assays for BAR and KR were very similar to their respective bases, with typical measuring ranges of 0.07 - 28 pmol (0.025 -10 ng) and 0.07 - 28.8 pmol (0.025 -10 ng), respectively. Purification ofplant material for RIA
Analysis of the ethanolic extract without prior Dowex purification was investigated initially because of the potential degradation of K during Dowex purification. However, the extract dilution curve did not parallel the standard curve, indicating the presence of contaminants not removed by TLC. These were removed with Dowex treatment, as indicated by parallelism between the extract dilution curve and standard curve. The method of Dowex purification used was chosen to reduce the risk of localized temperature and pH extremes generally associated with the use of a strong cation exchange resin column (Dyson and Hall, 1972). The degradation of 14C_K to adenine using this Dowex procedure was examined, and found to be negligible.
J Plant Physiol. Vol. 124. pp. 289-298 (1986)
292
P. J. HOFMAN, CLARE FORSYTH and J. VAN STADEN
7 6
3 2
o
0- - - -0- - - 0- - - - - -0 - - - - - - - - - - - -0
6
12
24 TIME hours
48
Fig. 2: Mass of K (--e--) and KR (- - - - -0- - - - -) detected in 20 ml culture medium containing 5 g soybean callus at various times of incubation after the addition of 14C-K, as determined by RIA. Bars indicate variation between duplicate samples. Where there are no bars, the variation was smaller than the symbol.
Metabolism ofK by soybean callus Analysis by RIA indicated an 80 % reduction in detectable K during the initial 24 h of incubation (Fig. 2). In the following 24 h period, no further decrease occurred. Kinetin riboside was not detected at any time during the 48 h incubation period. The production of potentially cross-reacting 3- and 9-glucosides (Weiler and Spanier, 1981) was investigated by assaying all Rfs from TLC by RIA. Significant additional assay response was obtained at Rf 0.2-0.25 only (Fig. 3). This corresponded to an intense UV-quenching spot at Rf 0.20-0.23, with consistent intensity throughout the incubation period. This was in contrast to a faint UV-quenching spot at Rf 0.77 0.82 (corresponding to K) which decreased in intensity with longer incubation. Thus the compound at Rf 0.20 - 0.23 was not considered to represent a cross-reacting K metabolite. Analysis by conventional chromatographic techniques (Sephadex LH-20 and HPLC) indicated that 14C_K was rapidly metabolised, and Sephadex LH-20 chromatography revealed that a number of metabolic products appeared as a result (Fig. 4). Each radioactive peak was further analysed by HPLC. No evidence was found to in-
J. Plant Physiol. Vol.
124. pp. 289-298 (1986)
Metabolism of kinetin and benzyladenine
KR
293
K
g2 Cf)
I-
Z
W .....I
a.~
1.0
w
:lI:
0 0.5
0
1.0
Rf Fig. 3: The immunoreactivity (expressed as K equivalents) of various Rfs from TLC of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 24 h after 14C_K was applied. Bars indicate Rfs at which visible UV-quenching occurred. K = kinetin, KR = ribosylkinetin.
Ado
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~
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6
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0
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4
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800
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1200
1600
ElUTION VOLUME ml Fig. 4: Sephadex LH-20 separation of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 24 h after 14C-K was applied. The column was eluted with 35 % ethanol. Ade = adenine, Ado = adenosine, K = kinetin.
J Plant Physiol. Vol. 124. pp. 289-298 {1986}
15
5
cr' ,.cr - -.().. - - - - -0- - - - - - - - - - - - -
o
0
48 Fig. 5: Mass of BA (--e--) and BAR (- - - - -0- - - - -) detected in 20 ml culture medium containing 5 g soybean callus at various times of incubation after the addition of 14C-BA, as determined by RIA. Bars indicate variation between duplicate samples. Where there are no bars, the variation was smaller than the symbol.
BAR
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Fig. 6: The immunoreactivity (expressed as BA equivalents) of various Rfs from TLC of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 24 h after 14C_BA was applied. Bars indicate Rfs at which visible UV-quenching occurred. BA = benzyladenine, BAR = ribosylbenzyladenine.
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295
BA
o
o
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1200
800
1600
ELUTION VOLUME ml
Fig. 7: Sephadex LH-20 separation of an extract of 5 g soybean callus plus 20 ml culture medium which had been incubated for 12 h after 14C_BA was applied. The column was eluted with 35 % ethanol. BA = benzyladenine, Ade = adenine, Ado = adenosine.
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J. Plant Physiol.
Vol. 124. pp. 289-298 (1986)
P.]. HOFMAN, CLARE FORSYTH and]. VAN STADEN
296
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Fig. 9: A. HPLC separation of authentic standards (--) and the radioactive peak (- - - - -e- - - --) which, on Sephadex LH-20, had an elution volume of 1240 -1360 ml. See Materials and Methods for the conditions used. B. As for A except that the peak having an elution volume of 960 -1040 ml (Fig. 7) was analysed. Ade = adenine, BA = benzyladenine, BAR = ribosylbenzyladenine.
dicate the presence of KR during the experimental period. The percentage radioactivity associated with K declined from 73.2 % at the start to 9.2 % after 24 h, and then to 0.62 % after 48 h of incubation. Other metabolic products were formed and, although these were further analysed, they are reported on in a concurrent paper (Forsyth and Van Staden, 1986), as the aim of this paper was to compare RIA with more conventional assay procedures. None of these metabolites were of the glucosylated or phosphorylated nature, and the only ribosylated derivative detected was adenosine.
Metabolism of BA by soybean callus Analysis by RIA indicated that BA also underwent a rapid metabolism (Fig. 5). However, in contrast to K, a rapid decrease in detectable BA occurred during the first 12 h, which continued at a reduced rate during the following 36 h. At 48 h a 92 % reduction in BA was observed. BAR was first detected at 6 h, reached a maximum at 12 h, and decreased by 78 % at 48 h. An analysis of the TLC plate revealed the presence of BA and BAR, but, as with K, no other cross-reacting compounds likely to correspond to BA glucosides were detected (Fig. 6). Analysis by conventional chromatography revealed that the rate of metabolism of 14C_BA was similar to that of 14C_K, and after only 12 h incubation a number of metabolites were present (Fig. 7). However, the percentage radioactivity associated with
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Vol. 124. pp. 289-298 (1986)
Metabolism of kinetin and benzyladenine
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BA declined during the first 6h of incubation from 82.4% to 25.9%. Thereafter the decline was slower until, at 48 h, only 10.3 % of the tot;li radioactivity was associateJ with BA (Fig. 8 and Fig. 9 A). HPLC analyses revealed that small amounts of BAR were formed and first appeared after 12 h incubation (7.2 % of the total recovered radioactivity). Thereafter the levels declined marginally to 3.5 % after 48 h incubation (Fig. 8 and Fig. 9 B). Again other metabolites were detected and are reported on in a concurrent paper (Forsyth and Van Staden, 1986). No metabolite apart from adenosine responded to acid treatment (Dyson et al., 1972), indicating that none of these products were of a glucosylated, ribosylated or phosphorylated nature. Discussion Neither the antiserum nor the TLC system used had the ability to distinguish between K and DHZ, and their ribosides. However, soybean callus does not inherently contain DHZ or DHZR (Van Staden and Davey, 1977), and thus does not possess the capacity for their synthesis. It is, therefore, highly improbable that soybean callus has the capacity to synthesise DHZ from added K. Therefore the TLC system used was considered adequate for this investigation, despite the fact that K (Rf 0.6) and KR (Rf 0.46) chromatographed very closely with DHZ (Rf 0.66) and DHZR (Rf 0.44). If K analyses are to be conducted on plant tissue likely to contain the dihydro-derivatives, a TLC system which adequately separates these compounds, such as silica gel developed with chloroform: methanol (9 : 2) could be employed. The assays developed proved to be a rapid means of assaying K, BA, and their ribosides. Assay sensitivity was similar to those developed previously (Hofman et al., 1985,1986), despite the fact that the antiserum used was not produced specifically for these synthetic cytokinins. Little sample purification was required. The agreement between the results obtained by RIA and conventional Sephadex LH-20 and HPLC was good. More metabolites were detected when Sephadex LH-20 and HPLC techniques were employed (Forsyth and Van Staden, 1986), not because of the relative sensitivity of the two techniques, but primarily because of the specificity of the antiserum used (Hofman et al., 1985). This is the major drawback of the use of RIA in metabolic studies, and if the detection of metabolites, in addition to the metabolism of the initial compound is required, then additional less specific techniques will be needed. Acknowledgements The financial assistance of the C.S.I.R., Pretoria and the University of Natal Research Fund is gratefully acknowledged.
References ARMSTRONG, D. J., W. J. BURROWS, P. F. EVANS, and F. SKOOG: Isolation of cytokinins from tRNA. Biochem. Biophys. Res. Commun. 37, 451-456 {1969}. DYSON, W. H. and R. H. HAu.: N 6-(L>2-isopentenyl}adenosine: Its occurrence as a free nucleoside in an autonomous strain of tobacco tissue. Plant Physiol. 50, 616-621 {1972}. DYSON, W. H., J. E. Fox, and J. D. MCCHESNEY: Short term metabolism of urea and purine cytokinins. Plant Physiol. 49, 506-513 {1972}.
J. Plant PhysioL VoL 124. pp. 289-298 (1986)
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FORSYTH, C. andJ. VAN STADEN: The metabolism and cell division activity of adenine derivatives • in soybean callus. J. Plant Physiol. 124,275-287 (1986). HOFMAN, P. J., C. FoRSYTH, andJ. VAN STADEN: A radioimmunoassay for dihydrozeatin and dihydrozeatinriboside, and its application to a study of the in vitro metabolism of dihydrozeatin by soybean callus. J. Plant Physiol. 121, 1-12 (1985 a). HOFMAN, P.J., B. C. FEATONBy-SMITII, andJ. VAN STADEN: The development of ELISA and RIA for cytokinin estimation and their application to a study of lunar periodicity in Ecklonia maxima (Osbeck) Papenf. J. Plant Physiol. 122, 455-466 (1986). VAN STADEN, J. and J. E. DAVEY: The metabolism of zeatin and zeatin riboside in soya bean callus. Ann. Bot. 41,1041-1048 (1977). WEILER, E. W.: Plant hormone immunoassay. Physiol. Plant. 54, 230-234 (1982). WEILER, E. W. and K. SPANIER: Phytohormones in the formation of crown gall tumors. Planta 153,326-337 (1981). WHENHAM, R. J.: Evaluation of selective detectors for the rapid and sensitive gas chromatographic assay of cytokinins, and application to the analysis of cytokinins in plant extracts. Planta 157, 554-560 (1983).
J. Plant Physiol. Vol. 124. pp. 289-298 (1986)