Long Term Metabolism of Zeatin Supplied to Soybean Callus

Long Term Metabolism of Zeatin Supplied to Soybean Callus J. VAN STADEN Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa Received May 28,1983 . Accepted June 10, 1983

Summary The biologically most-active metabolite formed when soybean callus was cultured in the presence of zeatin for 28 days was a conjugated compound which could be hydrolysed partly by (Jglucosidase. From the data obtained with labelled zeatin it was, however, evident that only a small proportion of the radioactivity recovered was associated with this metabolite. Most of the recovered radioactivity was associated with a compound which had similar chromatographic properties to N-(purin-6-yl)glycine, an oxidation product of zeatin, which is not very active in the soybean callus bioassay. This indicates that, while some glucosylated cytokinins accumulated in the callus, most of the applied zeatin was converted to other compounds which were either related to the inactivation or utilisation of the applied cytokinin.

Key words: Glycine max, metabolism, zeatin, soybean callus.

Introduction Both unlabelled (Van Staden and Davey, 1977) and labelled zeatin (Horgan, 1975; Van Staden and Hutton, 1982; Van Staden, 1983) has been applied to soybean callus in attempts to establish its fate and possible function during the process of cell division. In most of these studies, which were conducted over the short periods of 24 and 48 h, compounds which co-eluted with cytokinin glucosides, and which were susceptible to (1-glucosidase hydrolysis, appeared to be the major metabolites formed. The production of these glucosides apparently involves secondary steps in cytokinin metabolism (Van Staden and Hutton, 1982) relating to their inactivation (Horgan, 1975), or the production of storage compounds (Parker and Letham, 1973). Should this be the case then an accumulation of cytokinin glucosides should be detected in long term experiments when optimal levels of cytokinin are fed to callus tissues. The validity of this hypothesis was investigated by feeding zeatin to soybean callus for 28 days.

Materials and Methods Cr.0kinin requiring callus of Glycine max cv. Acme was cultured on a medium containing 10- M zeatin for 28 days at 26 ± 1 0c. The callus was cultured either on unlabelled zeatin (10- 5 M) "spiked" with 8[14C]zeatin (specific activity 441.1 MBq mmol- 1) to the extent that 1 ml of the final culture medium contained about 10,000 dpm. After autoclaving the medium, small pieces of soybean callus were transferred aseptically to the culture flasks. The experiment was terminated after 28 days and both the callus and agar deep-frozen for later analysis.

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Cytokinins were extracted, and the biological- and radioactivity determined as described previously (Van Staden, 1981). Extracts purified through Dowex 50 were separated on paper using iso-propanol: 25 % N~OH: water (10: 1 : 1 v/v), or they were fractionated on a Sephadex LH20 column with 10% methanol as eluant (Hutton and Van Staden, 1981) both before and after iJ-glucosidase treatment (Smith and Van Staden, 1978). -

Results and Discussion After 28 days of culturing on a medium which contained 10-5 M zeatin two peaks of biological activity were detected in the soybean callus bioassay (Fig. 1 A). The first peak (peak 1) was of a polar nature and occurred at Rf 0.2-0.3. The second peak (peak 2), which was responsible for most of the detected cytokinin-like activity, occurred at

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Rf 0.5--0.7, and co-chromatographed with zeatin and ribosylzeatin. Two peaks of radioactivity were also detected when the callus was cultured on a medium containing labelled zeatin (Fig. 1 B). These peaks had similar Rf values to those of the biologically active peaks detected when unlabelled zeatin was used as a cell division inducing factor (Fig. 1 A). Most of the radioactivity recovered (56.3 %) was associated with the polar peak (peak 1) which was responsible for only a small proportion of the detected biological activity. Only 13.7 % of the radioactivity recovered from the callus co-chromatographed with zeatin. Two peaks of radioactivity were also detected in the culture medium (agar) on which the callus had been growing for 28 days (Fig. 1 C). Most of the radioactivity recovered (78.8 %) from the medium coZ. Pjlanzenphysiol. Rd. 111. S. 423-428.1983.

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chromatographed with authentic zeatin. A polar peak of radioactivity (peak 1) which was responsible for 11.5 % of the radioactivity recovered was also detected in the culture medium. An analysis of the radioactivity detected in the Dowex 50 extracts of both the callus and the agar revealed that of the total radioactivity recovered 83.5 % was still in the culture medium and only 16.5 % in the soybean callus. These results indicate that there was a slow, regulated uptake of cytokinin by the callus and that a large proportion of what was taken up was metabolised to polar compounds. As the polar peak of radioactivity was also detected in the culture medium it seems reasonable to assume that the labelled products formed, were released readily into the medium. Elution and subsequent fractionation of peak 1 (Rf 0.1-0.4; Fig.l) on a Sephadex LH-20 column using 10 % methanol resulted in the detection of a number of radioactive and biologically active peaks (Fig. 2 A). The major radioactive peak co-eluted

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Fig. 3: Separation of authentic cold (UV-trace) and radioactive (e--e) purine derivatives (A), and cytokinin extracts obtained from soybean callus following the addition of 8(14C]zeatin to the culture medium (B and q, by reversed-phase HPLC. Column Micropak MCH-5 (5 ILm, C18 bonded, 150x4mm I.D.); flow rate lmlmin- 1; mobile phase, water to 4% acetonitrile over 10 mins, then to 30 % acetonitrile over 20 min. Absorbance was recorded with a Varian variable wavelength monitor at 265 nm which was fitted with a 8 ILl flow-through cell. Separation was achievep using a Varian 5000 Liquid Chromatograph. B = Aliquot (10 ILl) of the radioactive peak which co-eluted with N-(purin-6-yl)glycine (Fig. 2A and B). C = Aliquot (10 ILl) of the peak which after /3-glucosidase treatment co-eluted with zeatin (Fig. 2 B). Abbreviations as in Fig. 2.

with N-{purin-6-yl) glycine (elution volume 28~00 ml) both before (Fig. 2 A) and after J3-glucosidase treatment. No biological activity was associated with this peak. When subjected to HPLC analysis a radioactive peak which co-chromatographed with N-{purin-6-yl)glycine was detected (Fig. 3 B). The two major biologically active peaks, and which contained radioactive compounds, eluted between 480-560 ml and 600-680 ml respectively (Fig. 2 A). Following J3-glucosidase treatment the radioactivity and biological activity associated with these two peaks decreased (Fig. 2 B). Particularly noticeable was that there was subsequently an increase in radioactivity and biological activity at the elution volume where zeatin is normally detected. Following fractionation of this latter peak by HPLC only one radioactive peak was detected which had the same retention time as trans-zeatin (Fig. 3 C). The fourth radioactive peak (elution volume 960-1000 ml) detected after Sephadex LH-20 fractionation cochromatographed with adenine and was not affected by J3-glucosidase treatment (Fig. 2 A and B). The non-polar peak detected on paper (Rf 0.5-0.7; Fig. 1) yielded two peaks of biological activity following Sephadex LH-20 fractionation (Fig. 2 C). Both of these Z. Pjlanzenphysiol. Ed. 111. S. 423-428. 1983.

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peaks, which co-eluted with ribosylzeatin and zeatin respectively, were associated with radioactivity. Following separation by HPLC the radioactivity which was associated with ribosylzeatin co-chromatographed with trans-ribosylzeatin (Fig. 4 B). The peak which co-chromatographed with zeatin (Fig. 2 C) had a similar retention time as trans-zeatin (Fig. 4 C).

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Fig. 4: Separation of authentic cold (UV-trace) purine derivatives (A), and radioactive (e--e) cytokinin extracts obtained from soybean callus following the addition of S[I4C]zeatin to the culture medium (B and C), by reversed-phase HPLC. Column Micropak MCH-5 (5fLm, CIS bonded, 150x4mm LD.); flow rate 1 ml min-I; mobile phase, water (0.2M acetic acid) to 4% acetonitrile (0.2M acetic acid) over 5 min, isocratically for further 10 min, then to S % acetonitrile over 10 min. Conditions otherwise as in Fig. 3. B = Aliquot (10 fLI) of the radioactive peak which co-eluted with ribosylzeatin (Fig. 2 C). The extract was «spiked» with authentic ribosylzeatin. C = Aliquot (10 fLl) of the radioactive peak «
Short term experiments (Van Staden and Hutton, 1928; Van Staden, 1983) have indicated that zeatin applied direct onto soybean callus was rapidly metabolised by means of ribosylation, glucosylation, and reduction and oxidation reactions. In this relatively long term investigation evidence was obtained that the biologically most active metabolites present when soybean callus was grown on a medium containing unlabelled or labelled zeatin were conjugated compounds which were partly hydrolysed by {3-glucosidase. This suggests that the products which do accumulate, and which are active in the soybean callus bioassay are cytokinin glucosides, as was found in the short term (Horgan, 1975). From the radioactive data it is however, evident that the largest proportion of the labelled zeatin taken up by the callus was metabolised to a polar compound which co-eluted with N-{purin-6-yl)glycine both on a Sephadex LH-20 column and after reversed-phase HPLC. This provides circumZ. Pjlanzenphysiol. Ed. 111. S. 423-428. 1983.

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stantial evidence for the inactivation of that part of the applied zeatin which was oxidized, particularly as the end product is not very active in the soybean callus bioassay (Van Staden and Drewes, 1983). Whether this metabolic step forms part of the utilization process of the applied cytokinin is not known. Using labelled zeatin, the earlier finding (Van Staden and Davey, 1977) that a proportion of the applied zeatin is ribosylated, was corroborated. Acknowledgements Financial support was received from the C.S.1.R. and Atomic Energy Board, Pretoria.

References HORGAN, R.: A new cytokinin metabolite. Biochem. Biophys. Res. Commun. 65, 358-363 (1975). HUTTON, M. J. andJ. VAN STADEN: An efficient column chromatographic method for separating cytokinins. Ann. Bot. 47, 527-530 (1981). PARKER, C. W. and D. S. LETHAM: Regulators of cell division in plant tissues. XVI. Metabolism of zeatin by radish cotyledons and hypocotyls. Planta 114, 199-218 (1973). SMITH, A. R. andJ. VAN STADEN: Changes in endogenous cytokinin levels in kernels of Zea mays L. during imbibition and germination. J. Exp. Bot. 29, 1067-1075 (1978). VAN STADEN, J.: Cytokinins in germinating maize caryopses. 1. Transport and metabolism of 8[14C]t-zeatin applied to the endosperm. Physiol. Plant. 53, 269-274 (1981). - Short term metabolism of different concentrations of zeatin applied to soybean callus. Z. Pflanzenphysiol. 109, 163-169 (1983). VAN STADEN, J. and J. E. DAVEY: The metabolism of zeatin and zeatin riboside by soya bean callus. Ann. Bot. 41,1041-1048 (1977). VAN STADEN, J. and S. E. DREWES: Properties and activity of N-{purin-6-yl)glycine in the soya bean callus bioassay. Ann. Bot. 51,149-151 (1983). VAN STADEN, J. and M. J. HUTTON: Metabolism of 8[14C]t-zeatin in soybean callus. Z. Pflanzenphysiol. 106, 355-365 (1982).

Z. Pjlanzenphysiol. Ed. 111. S. 423-428. 1983.