Planta
Planta (Berl.) 132, 1 - 8 (1976)
9 by Springer-Verlag 1976
Cytokinins of Sycamore Spring Sap J.G. Purse*, R. Horgan, J.M. Horgan, and P.F. Wareing Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, U.K,
Summary. The cyt0kinins present in the spring sap of Acer pseudoplatanus L. were investigated. Ribosyltrans-zeatin, trans-zeatin and dihydrozeatin were isolated and identified by combined gas chromatography-mass spectrometry (GC-MS). A number of other cytokinin active fractions were observed. One of these was less polar than zeatin and did not behave as any known cytokinin. Two other fractions were more polar than ribosylzeatin and were unstable. A decomposition product of one of these was identified as ribosyl-trans-zeatin by GC-MS. The possible nature of the unstable compounds is discussed. Data on the change s in cytokinin activity of the various fractions during spring 1973 are presented and discussed.
Introduction
Most studies on cytokinins in plant tissues have yielded only limited information on the nature of the compounds present and the qualitative and quantitative changes in these compounds during plant development, due to the sole use of bioassays as the method of detection and estimation. The ability to identify all the cytokinins present in a tissue is a prerequisite for obtaining more detailed information on the action of these compounds. In certain plant tissues, cytokinins are present in sufficiently high concentrations to permit isolation of some of the compounds by crystallisation from purified extracts (e.g. Letham, 1963 ; Koshimizu et al., 1967). Approx. 70 I.tg is the minimum quantity of cytokinin that has been Present address: Milstead Laboratory of Chemical Enzymology, Broad Oak Road, Sitting, Kent, ME9 8AG, U.K. Abbreviations. GLC=gas-liquid chromatography; G C - M S = combined gas chromatography-mass spectrometry; KE=kinetin equivalents ; TLC = thin-layer chromatography; TMS = trimethylsilyl; tRNA=transfer RNA; i6Ade=6-(3-methylbut-2-enylamino)purine; i~Ado = 6-(3-methylbut-2-enylamino)-9-/?-D-ribofuranosylpurine *
obtained by this method (Parker and Letham, 1973). However, in many tissues of physiological interest cytokinins are apparently present in very low concentrations and it is usually impracticable to extract sufficient material to enable crystalline cytokinins to be obtained. We have therefore developed techniques to permit the isolation of between 1 and 10 lag of cytokinins from plant material and to identify these as their TMS derivatives by combined GC-MS. The preparation and GLC of TMS-cytokinins has been reported by Most et al. (1968) and the cytokinins of tRNA and bacterial cultures (Babcock and Morris, 1970; Upper et al., 1970) have been investigated using such procedures. However, such methods have only occasionally be used to investigate the free cytokinins of plants (Dyson and Hall, 1972; Yoshida and Oritani, 1972), mainly because of the considerable degree of prior purification required to permit successful analysis by GLC. We have used sycamore (Acer pseudoplatanus L.) spring sap as a source of cytokinins with which to develop the necessary purification procedures and we have aimed to use as mild chemical procedures as possible throughout the purification procedure. Sycamore sap has been shown to be a relatively rich source of cytokinins (Hewett, 1973). Our group has previously reported the identification of ribosyl-trans-zeatin and N6-(2-hydroxybenzyl)adenosine from sycamore sap (Horgan et al., 1973 a) and poplar leaves (Horgan et al., 1973b) respectively, using similar methods. We have also determined the changes in the amounts of cytokinins in fractions of sycamore sap obtained during spring 1973, as estimated by bioassay of the partially purified extracts. Materials and Methods Plant Material
500 1 of sycamore spring sap was collected from 15 mature, freestanding trees in the Botany Gardens, University College of Wales,
2 Aberystwyth between 23rd January and 16th April 1973, and 19th January and 14th February 1974. The sap was obtained by drilling a 25 m m diameter hole approximately 50 m m into the trunks 0.5 m above ground level, and inserting a greased cork through which was fitted a glass tube. The tube was connected to a 2.5 1 bottle containing 10 ml of toluene as bactericide. The sap was collected from these bottles daily for 6 days, after which new holes were drilled. After filtering, the sap was reduced to approximately onetenth its original volume in a cyclone evaporator at 25 ~ and further reduced to a thin syrup on a rotary evaporator, the water bath never exceeding 35~ The syrup (pH 5.5-6.0) was stored in batches in plastic bottles at - 2 0 ~ until required.
Chemicals and Solvents Analytical grade reagents were used where possible. Organic solvents were glass-distilled before use. Water used in later stages of extract purification was double-distilled in glass. Kinetin and i6Ade were obtained from Sigma Chemical Co. a n d i6Ado, zeatin, 9-/~-D-ribosylzeatin and (_+)-dihydrozeatin from Calbiochem. 6-chloro[814C]purine (10 mCi m m o l - x ) was obtained from the Radiochemical Centre, A m e r s h a m . The following cytokinins were synthesised in this laboratory by condensing 4-hydroxy-3-methylbut-trans-2enylamine with the material in brackets: trans-zeatin (6-chloropurine), 9-~-D-ribosyl-trans-zeatin (6-chloropurine-9-triacetyl-/~-Dribofuranosylpurine), trans-9-~-D-glucopyranosylzeatin (6-chloro9-tetra~acetyl-/~-D-glucopyranosyl-purine), trans-[8-14C]zeafin (6chloro[8-14C] purine).
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap in the former case and from recovered c p m in the latter. Zones with mobility similar to ribosylzeatin were eluted with E t O H : H 2 0 (4:1, v/v)for 48 h. Approximately 25% of a 5 gg sample of ribosyltrans-zeatin (estimated from G L C peak area measurements) could be recovered from a plate under these conditions. Such eluates also contained impurities arising from the plates which were detectable by G L C under the conditions used and greater proportions of water in the eluting solvent led to heavy contamination of samples.
GLC and GC-MS T M S derivativies were prepared by heating extracts or authentic c o m p o u n d s in 50 ~tl N,O-bis-trimethylsilyltrifluoracetamide:trimethylchlorosilane:hexamethyldisilazane (2:1:1, v/v) for 1 hr at 80 ~ in 0.5 ml vials. Pure N,O-bis-trimethylsilylacetamide was found to be an equally good reagent. Gas chromatography was performed on a Pye 104 fitted with dual flame ionization detectors and dual 1.65 m x 4 m m glass columns containing 2% OV-I on 100-120 mesh G a s C h r o m Q. Typical operating conditions were: carrier gas (Nz) 40 ml m i n - 1 ; H2 40 ml m i n - 1 ; air 600 ml m i n - ~ ; detectors 300 ~ oven temperature p r o g r a m m e d from 180 ~ to 300 ~ at 12 degrees m i n - 1 . Full-scale recorder deflection under these conditions was given by approximately 0.05 ~.g of TMS-cytokinin at an amplifier attenuation of 100. The G C - M S system was a Pye 104 coupled to an AEI MS30 mass spectrometer via an all-glass inlet and a silicone rubber m e m b r a n e separator. G L C conditions were the same as previously described, except that He was used as carrier gas and the mass spectrometer was used as detector. Spectra were recorded on photosensitive paper at an ionisation voltage of 24 eV.
Chromatographic Methods The following solvent systems were used: A - EtOH: HzO (92:8 v/v, containing 1 0 - 3 M HCI or CF3COOH); B - E t O H : H z O (7:13, v/v) ; C - CHC13 : M e O H (9 : 1 v/v) ; D - butan-2-ol: 12 M N H g O H (4:1 v/v). Sephadex LH-20 columns were eluted with solvents A and B. A column of LH-20 (Pharmacia, 1.8 m x 25 m m in a Chromatronix glass c o l u m n fitted with teflon bed supports) was continually run at 3 0 m l h -~ with solvent A at 2 . 1 x 1 0 4 k g m ~. The column was packed with LH-20 swollen in acetone and solvent A was forced t h r o u g h the column with a constant-flow Labotron pump. The gel expansion caused by replacement of acetone with solvent A caused a pressure increase which was maintained. Samples were injected onto the column through a septum-sealed injection port a n d up to 2 1 of eluate were collected in 50 ml or 60 ml fractions. A n o t h e r column of LH-20 (0.9 m x 25 m m , in a Chromatronix glass column fitted with teflon bed supports) was continually run at 30 ml h - 1 with solvent B. This column was packed in conventional m a n n e r and the flow was maintained continuously using a reciprocating p u m p (Metering P u m p s Ltd.). For each sample applied to the c o l u m n up to 3 1 of eluate were collected in 30 ml fractions. The recovery of [8-~4C] zeatin from both these c o l u m n s was 100% of that applied. T L C was carried out on pre-coated Merck silica gel PF2~4 plates (200 x 50 x 0.25 m m ) using solvent C (Playtis and Leonard, 1971). The plates were washed by continual development in EtOH: glacial acetic acid (4:1 v/v) for 48 h, followed by continual development with solvent C for 24 h. Extracts were c h r o m a t o g r a p h e d alongside cytokinin markers. To prevent any association of markers a n d sample the plates were scored lengthwise. Plates were continually developed until the zone thought to be of interest had migrated approx. 10 cm from the origin. Markers were located by viewing under a U V (254 nm) lamp. C h r o m a t o g r a m zones with mobility greater than or equal to trans-zeatin were eluted with 3 ml EtOH for 48 h. W h e n 5 l-tg each of i6Ado and trans-[8-~4C]zeatin were eluted under these conditions, the recoveries were 95% and 65% respectively. Recoveries were estimated from G L C peak areas
Bioassay Procedure The soybean callus assay (Miller, 1963) was used. Solutions to be assayed were placed in 100 ml conical flasks and dried on a hotplate at 40 ~ in a stream of air. 25 ml of medium was then added to the flasks, which were then autoclaved. The flasks were inoculated with three pieces of callus (approx. 0.05 g each). The cultures were grown at 26-27 ~ for three weeks under diffuse white light and the callus in each flask was then weighed. A duplicated set of kinetin controls was included in each assay. Growth of callus in an assayed fraction was expressed in gg kinetin equivalents (gg KE), this being the a m o u n t of kinetin required to elicit the same callus growth response as an active fraction in a particular assay.
Purification Procedure used on Sap Sap was thawed, adjusted to p H 3.1, filtered and percolated t h r o u g h a column of Zerolit 225 cation-exchange resin (Permutit, 52 100 mesh, 450 x 2 5 ram, N H + form, equilibrated at p H 3.1) at approx. 5 ml rain -~ (Letham, 1968). The column was washed with 1 litre of water and the effluents were combined to give fraction A. Basic substances were eluted from the column with 1 litre 1 M N H 4 O H to give fraction B. Fraction B was further purified by partitioning with organic solvents. After reduction to 500 ml the fraction was on some occasions partitioned against 500 ml EtOAc at p H 2.5. This procedure led to very slight losses of cytokinin activity from the aqueous phase (Fig. 5). The aqueous extract was partitioned against water-saturated n B u O H (4 x 500 ml) at p H 8.2. A second series of n B u O H extractions was carried out on an aqueous residue at this stage but very little cytokinin activity could be detected in it, following further purification. The n B u O H phase was reduced to dryness and dissolved in the m i n i m u m quantity of solvent D. This solution was loaded onto a cellulose column ( W h a t m a n C F l l , 240 x 20 m m , 10 g dry weight cellulose) and eluted with 350 ml solvent D, A small quan-
3
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap tity (1 gg) of trans-[8-14C]zeatin dissolved in water was taken through the procedure described so far, the ethyl acetate extraction being omitted. The overall recovery of the counts applied was 50%. The cellulose column eluate was reduced to dryness, redissolved in 2 ml solvent A and chromatographed on LH-20 in solvent A. A portion of each fraction collected was bioassayed, the remainder of each fraction being stored at - 2 0 ~ until the bioassay result was obtained. Fractions of interest were then reduced to dryness, re-dissolved in 1-2 ml solvent B and chromatographed on LH-20 in solvent B. A portion of each fraction collected was again bioassayed, the remainder being stored at - 20 ~ until required. Fractions required for analysis by G L C and GC-MS were either re-chromatographed on LH-20 in solvent B or chromatographed on silica gel in solvent C prior to silylation. All glassware used in the preparation of samples for G L C was cleaned with chromic acid. Fraction A was partitioned against n B u O H in the same manner as fraction B. The aqueous phases remaining after partitioning the two fractions were combined, reduced to 50 ml and any ribotides present were hydrolysed using alkaline phosphatase (orthophosphoric monoester phosphohydrolase [E.C.3.1.3.1 .], Sigma, calf intestinal mucosa). The solution contained 1 0 - 2 M MgC12 and incubation was for 24 h at 27 ~ p H 8. 1 ml toluene was added as bactericide. The solution was partitioned with 4 x 50 ml n B u O H and the combined organic phases were treated as the organic extract of fraction B.
Results
Identification of Ribosyl-Trans-Zeatin, Dihydrozeatin and Trans-Zeatin A 20 1 batch of sap collected 16th 17th February 1973 was purified as described in ,,Materials and Methods", the ethyl acetate extraction being omitted. Following chromatography on LH-20 in solvent A, a single peak of biological activity was observed at the elution volume of zeatin and ribosylzeatin. The remainder of this zone was chromatographed on LH-20 in solvent B, and cytokinin activity was observed in two incompletely resolved peaks at the elution volumes of ribosylzeatin and zeatin (Fig. 1. Zone A1, fraction 1718, approx. 7.5 gg K E 1-1 sap. Zone A2, fractions 20-22, approx. 50 ~tg K E 1- 1 sap). Fractions 2 3 and 24 (Fig. 1) were re-chromatographed in the same system and fractions collected were bioassayed. All biological activity eluted in Zone A2, indicating that the column had probably been over-loaded in the first run. Zones A1 and A2 were chromatographed separately on silica gel TLC plates in solvent C and zones from the plates were eluted. A portion of each eluate was bioassayed. On the plate containing zone A1, cytokinin activity was solely associated with a UV- absorbing band adjacent to the ribosyl-trans-zeatin marker. Assuming that all this band's absorbance was due to ribosylzeatin, it was estimated that approx. 8 ~tg of this compound was present. A UV-absorbing band was also observed adjacent to the ribosyl-cis-zeatin marker on
the plate, but no cytokinin activity was detected in the eluate of this zone. The remainder of each of the eluates corresponding to the two ribosylzeatin
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FRACTION NO.
Fig. 1. Cytokinin activity of fractions obtained following chromatography of a partially purified sycamore sap extract (20 1 sap, collected 16.2.73.-17.2.73.) on LH-20 in solvent B. 36 x 30 ml fractions were collected and a portion of each fraction equivalent to 1 1 of sap was assayed. Ribosylzeatin and zeatin markers elute in zones A1 and A2 respectively. Kinetin c o n t r o l s : - A , 0 ~tg 1-1; B10ggl 1;C100pgl 1;Dlmgl -I
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6 12 RETENTION TIME (min) Fig. 2. G L C trace of purified sycamore sap obtained from zone A1 (Fig. 1). A portion of A1 equivalent to 14 1 of sap was chromatographed on silica gel in solvent C and the UV-absorbing band adjacent to the ribosyl-trans-zeatin marker was eluted. One-fourteenth of this eluate was bioassayed, and the remainder was dried and silylated. 2 gl aliquots of this were used for G L C analysis. Dotted lines show the result of co-injecting an aliquot of sample with 0.1 gg trans-ribosylzeatin derivatised under the same conditions. Peaks Y and Z are due to TMSs-ribosyl-trans-zeatin and TMS4-ribosyl-trans-zeatin respectively. Attenuation was 5 x 102. The TMS4-trans-ribosylzeatin peak in the extract represents approximately 0.07 gg of material injected
4
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap F I
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Fig. 3. Cytokinin activity in a portion of zone A2 (Fig. 1), following chromatography on silica gel in solvent C and elution o f zones from the plate. Extract equivalent to 18 1 of sap was chromatographed and one-eighteenth of each eluate was assayed. A transzeatin marker spot migrated to zone E. O = origin, S = solvent front. Kinetincontrols:A0ggl 1;B 10/tgl-1; C 100ggl-1;D ling 1-1
la3
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RETENTION
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Fig. 4a and b. GLC traces of purified sycamore sap obtained from zone E (Fig. 3). A portion of the eluate of zone E was derivatised and aliquots of this solution were analysed by GLC. Attenuation was 20 x 102. a 3 gl aliquot of sample. Dotted line shows result of co-injecting such a sample with authentic TMS2-dihydrozeatin. b 2 •1 aliquot of sample. Dotted line shows result of co-injecting such a sample with authentic TMS2- and TMS3-trans-zeatin
isomers were silylated and a portion of each solution was gas chromatographed. In the eluate corresponding to the ribosyl-transzeatin marker a peak with the retention time of TMS4-ribosyl-trans-zeatin was observed. Bioassay of fractions of the extract collected during a preparative GLC run showed cytokinin activity only with the retention times of TMS4- and TMSs-ribosyl-transzeatin. When an aliquot of silylated extract was coinjected with authentic silylated ribosyl-trans-zeatin the peaks co-eluted (Fig. 2). Based on GLC peak area measurements, approximately 1.75 gg ribosyl-transzeatin was present in the silylated extract. Mass spec-
tra of this peak and of adjacent peaks were recorded during a GC-MS run with half the silylated material. The mass spectrum of the peak with the retention time of TMS4-ribosyl-trans-zeatin contained prominent ions at m/e 640, 639, 626, 625, 624, 551, 550, 549, 538, 537, 536, 321,320, 259, 245, 243, 230, 217, 202, 201,200, 188, 156, 147 and 103 which, by comparison with the spectrum of the authentic compound, were attributable to TMS4-ribosylzeatin. The spectrum also contained less prominent ions attributable to column bleed and preceding and succeeding peaks. All the mass spectra obtained were also examined for the presence of silylated dihydrozeatin riboside since, by analogy with dihydrozeatin and trans-zeatin, ribosyl-trans-zeatin and dihydrozeatin riboside would not be expected to separate in the chromatographic systems used prior to GLC. No evidence for its presence was found. When the eluate of the band corresponding to the ribosyl-cis-zeatin TLC marker was silylated and gas chromatographed a peak was observed with the retention time of TMS4-ribosyl-cis-zeatin, but a mass spectrum of this peak taken during a GC-MS run revealed no ions attributable to this compound. On the TLC plate containing zone A2, cytokinin activity was mainly associated with a UV-absorbing band adjacent to the trans-zeatin marker (Fig. 3, Zone E, approx. 40 Ixg KE 1 t sap) which, if entirely due to trans-zeatin was estimated to represent 1520 gg of material. A small amount of cytokinin activity of lower chromatographic mobility was also observed (Fig. 3. Zone F) and could be repeatedly demonstrated in this fraction. This zone was not further investigated, but the activity in it may have been due to a small quantity of ribosyl-trans-zeatin in zone A2. No cytokinin activity in the extract was associated with the cis-zeatin marker. The remainder of the eluate of zone C was silylated and a 2 lal portion was gas chromatographed. The only peaks observed were shown to co-elute with TMS/-dihydrozeatin, TMS3-zeatin and TMS2-zeatin when further portions of extract were co-injected with authentic compounds (Fig. 4). Bioassay of fractions collected during a preparative GLC run with a further portion of extract showed cytokinin activity to be associated with this group of peaks only. Mass spectral scans recorded during two GC-MS runs with further portions of the extract confirmed the presence of these compounds. The mass spectrum of the peak with the retention time of TMS2-dihydrozeatin contained ions at m/e 365, 351, 350, 292, 276, 275, 264, 262, 260, 235, 234, 221,220, 207, 192, 162, 148 and 135, indicative of the presence of this compound. The mass spectrum on the shoulder of the above peak, co-eluting with TMS3-trans-zeatin, contained ions at
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap AI
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Fig. Sa and b. Cytokinin activity in fractions obtained following chromatography of a ethyl acetate extract and b n-butanol extract of partially purified sycamore sap (30 1, collected 1st Feb.-3rd Feb. 1973) on LH-20 in solvent B. 3 0 x 3 0 ml fractions were collected and the equivalent of 1 1 of sap from each fraction was assayed. Zones A1, A2 and A3 correspond to the elution positions of ribosylzeatin, zeatin and i6Ado respectively. Kinetin controls: A0ggl 1;BlOp.gl-1;ClOOiagl-l;Dlmgl 1
m/e 436, 435, 434, 422, 421, 420, 348, 347, 346, 345, 332, 306, 305, 304 and 272, attributable to TMS3zeatin, together with ions corresponding to TMSzdihydrozeatin. The mass spectrum of the peak with the retention time of TMS2-trans-zeatin contained ions at m/e 363, 349, 348, 274, 273, 272, 261, 260, 258, 232, 201, 160 and 146, indicating the presence of this compound, together with other ions attributable to TMSa-dihydrozeatin. The amounts of transzeatin and dihydrozeatin thus isolated and identified were estimated by GLC peak size to represent 0.3 gg 1- ~ sap and 0.5 pg 1- 1 sap respectively. These figures take no account of losses of these compounds incurred during the purification procedure. No cytokinin activity was detected in any sap which was examined for the possible presence of cytokinin ribotides as described in "Materials and Methods". Investigation of a Further Cytokinin-active Fraction Present in two Batches of Sap A zone of cytokinin activity (zone A3 on the LH-20 column eluted with solvent B) was observed in two batches of sap collected lst-3rd February 1973 (batch I) and 6th-9th February 1973 (batch 2). It could not be detected in other batches collected in 1973-74 in spite of considerable investigation. Both
Fig. 6. Cytokinin activity in a portion of zone A3 (Fig. 5 b, equivalent to three litres of sap), following chromatography on silica gel in solvent C and elution of zones from the plate, Bars G and H indicate positions of i6Ado and i6Ade markers respectively. O=origin, S=solvent front. Kinetin controls: A 0 pg 1 1; B 10 pg 1 1; C 1 0 0 g g l - 1
batches were purified according to the standard procedure, the ethyl acetate extraction being included for batch 1. The ethyl acetate and butanol extracts of this batch were chromatographed separately on LH-20 in solvent B and a portion of each fraction collected was bioassayed (Fig. 5). Very little of the cytokinin activity present in the extract passed into the organic phase during the partitioning with ethyl acetate. However, slight callus growth was observed in zone A3 of this fraction and cytokinin activity could consistently be demonstrated in it throughout its subsequent purification. A portion of zone A3 obtained from the butanol fraction was chromatographed on a silica gel TLC plate in solvent C alongside i6Ade and i6Ado markers. Ten zones from the plate were eluted and a portion of each eluate was bioassayed. The only zone of biological activity observed was more mobile than both markers and co-incided with the only UV-absorbing spot in the extract (Fig. 6). Further details of the isolation and identification of this compound will be presented in a separate paper. Unstable Fractions Observed in Extracts In some batches of sap which were subjected to the normal purification procedure, cytokinin activity was observed in fractions collected from the LH-20 column eluted with solvent B at and before the elution volume of 9-glucosylzeatin (zones A4 and A5 respectively). In some such batches these fractions contained almost all the cytokinin activity in the extract, while in others they represented only part of the total cytokinin activity. When these fractions were re-chromatographed in the same system, cytokinin activity was usually observed at the elution volumes of ribosylzea-
6
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap A5 I
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Fig. 7. a Cytokinin activity in an extract of sycamore sap (45 1, collected 30th Jan.-3rd Feb. 1974), folIowing chromatography on LH-20 in solvent B. 24 x 30 ml fractions were collected and a portion of each fraction equivalent to 1 1 of sap was assayed, b The remainder of each fraction from a was stored for four weeks at - 2 0 ~ and fractions 10-12 were then re-chromatographed in the same system. 28 x 30 ml fractions were collected and portions of each fraction equivalent to 1 1 of sap were bioassayed. 9-glucosylzeatin, ribosylzeatin and zeatin elute in zones A4, A1 and A2 respectively. Kinetin controls A 0 lag 1- ~ ; B 10 lag 1 ~ ; C 100 lag 1-1
Ld U3 Z Iii rY
kW rm
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12 RETENTION
TIME (rnin)
Fig. 8. G L C trace of silylated material obtained from partially purified sycamore sap fractions containing the active decomposition product of an unstable cytokinin-active fraction (Fig. 7 b, fractions 19-20). The dried material was silylated and a 2 lal aliquot was injected into the gas chromatograph. The dashed line indicates the result of co-injecting a further 2 gl aliquot with 0.02 lag trans-ribosylzeatin derivatised in the same manner. Attenuation 2 x 102
tin and/or zeatin (Fig. 7), although in some cases all or part of the cytokinin activity revealed in the earliereluting zone. A batch of sap (45 1, collected 30th January-3rd February 1974) was purified in the usual manner,
the ethyl acetate extraction being omitted. Following chromatography on LH-20 in solvent B, cytokinin activity with an elution volume of 270-420 ml was observed (Fig. 7a). Following storage in solvent B at - 2 0 ~ for 4 weeks, fractions 10-12 (elution volume 270-360 ml) were re-chromatographed on LH-20 in solvent B, portions of each fraction collected being bioassayed. Cytokinin activity was observed mainly at the elution volume of ribosylzeatin (Fig. 7b, fractions 19 20). The remainder of fractions 19 and 20 were silylated. Gas chromatography of a portion of the silylated fractions gave a peak which co-chromatographed with authentic TMS4-ribosyl-trans-zeatin (Fig. 8), and the presence of this compound was confirmed with a mass spectral scan of this peak taken during a GC-MS run with the remainder of the sampie. The mass spectrum obtained contained prominent ions at m/e 639, 624, 550, 549, 536, 320, 276, 274, 260, 259, 245, 243, 230, 217, 204, 201, 200, 188, 156, 147 and 103 at very similar relative intensities to the authentic compound. Thus the principal cytokinin active decomposition product obtained from the unstable fraction was identified as ribosyl-trans-zeatin. The similar behaviour of the other unstable fractions observed suggested that they all decomposed to give either zeatin or ribosylzeatin. The order of elution of cytokinins from the LH-20 column eluted with solvent B closely parallel the partition coefficients of cytokinins between nBuOH and aqueous solutions at pH 7 or 8 (Letham, 1974; Purse, 1975) when the partition coefficients are arranged in increasing order, suggesting that this column operated mainly be reverse-phase partition rather than by adsorption as has previously been stated (Armstrong etal., 1969). Thus, in general, polar compounds would be expected to elute more rapidly from this column than less polar ones. Thus the compounds in the unstable active fractions were probably more polar than their active decomposition products. It is possible that these early-eluting fractions contained naturally-occurring, polar cytokinins or else were artefacts of the sap collection and/or purification procedure. In the latter case, such artefacts could be due to the formation of compounds more polar than ribosylzeatin or could be due to an association of a physical nature, e.g. association with another compound during chromatography. While insufficient evidence was available to distinguish between these possibilities, the evidence suggested that they were artefacts of some kind, since there is no obvious reason why unstable compounds should survive all the purification procedure prior to chromatography on LH-20 and yet decompose in aqueous ethanol at - 20 ~ Also, when a second series of butanol extraction was performed on the basic etuate from the ca-
J.G. Purse et al. : Cytokininsof SycamoreSpring Sap tion-exchange column, the extract contained very little cytokinin activity, even though if polar cytokinins had been present they would have incompletely partitioned into butanol during the first series of extractions, so that the butanol from the second series of extractions would also contain such cytokinins. A type of reaction which could lead to polar derivatives of zeatin and ribosylzeatin is reaction of a primary or secondary amine with a reducing sugar to yield an aldosamine or ketosamine (Reynolds, 1963, 1965). This type of reaction is said to occur in many syrups and freeze-dried foods and proceeds under mild conditions. The products formed are readily hydrolysed and always yield the parent amine. Attempts were made to carry out such a reaction by (a) adding 5 gg of authentic ribosyl-trans-zeatin to 1 1 of freshly collected sycamore sap, taking to dryness in vacuo and extracting the cytokinins by cation-exchange, butanol partition and chromatography on LH-20 in solvent B, (b) warming trans-[8-14C]zeatin with glucose or fructose under conditions similar to those used during the purification procedure, immediately followed by chromatography on LH-20 in solvent B. In neither case was any active material detected at elution volumes other than those of ribosylzeatin and zeatin respectively. Thus the nature of the cytokinin activity observed in these fractions remains to be elucidated.
Changes in Cytok&in Content of Sap Collected in Spring 1973 Figure 9 shows the changes in cytokinin activity observed in zones A1, A2, A 4 + A 5 and A1 + A 4 + A 5 during spring 1973. All points on these graphs are estimates of the total biological activity in the appropriate zone(s) from the LH-20 column eluted with solvent B, following assay of extract equivalent to 1 1 of sap. Sap was collected either on the indicated date or over a period of several days, in which case the date in the middle of this period is shown on the graphs. Sap was extracted according to the complete extraction procedure, except when only 1 2 1 of sap was being processed, when it was purified by cation-exchange , butanol partition and chromatography on LH-20 in solvent B only. Bud swelling on the trees was apparent in early March and budburst occurred around 20th March. No activity was detectable in zone A2 from sap collected in late January, but the activity in this zone reached a maximum in mid-February and subsequently declined somewhat. Both trans-zeatin and dihydrozeatin were identified in the sap at the time when it contained maximum activity, and dihydrozeatin was present in greater amounts at this time. However,
7
o 12 __
A2
oJ S t ~l~176
A,
1
O j
i
JAN.
i
FEB.
i
MAR.
APR,
Fig. 9. Changes in cytokinin activity in zones A2, AI, A4+A5 and A1 +A4+A5 of sycamore sap extracts during spring 1973, estimated from the cytokinin activity in fractions obtained from extracts chromatographedon LH-20 in solvent B since dihydrozeatin possesses only 10-20% of the cytokinin activity of trans-zeatin in the soybean assay, most of the activity shown at this point on the plot is presumably due to trans-zeatin. For reasons discussed above, it is probable that zones A4 and A5 are artefacts derived mainly from ribosylzeatin and probably zeatin also or are unstable, naturally-occurring derivatives of these compounds. Thus the principal active compound detected in bioassays of these zones may be ribosylzeatin itself. While the cytokinin activity detected in zones A1 and A 4 + A 5 fluctuated considerably during spring 1973, the total activity in these zones remained remarkably constant, apart from a peak in early February. Discussion
Previous work has indicated the presence of cytokinins in the spring sap of Acer pseudoplatanus (Reid and Burrows, 1968; Hewett, 1973) and our group has already reported the identification of ribosyltrans-zeatin from this source by GC-MS. The purification techniques have now been refined sufficiently to permit the separation, isolation and identification of further cytokinins. In the present work, the isolation and identification of ribosyl-trans-zeatin, transzeatin and dihydrozeatin using such techniques are described. Details of the characterisation of the cytokinin-active compound in zone A3 will be published separately. Unstable, cytokinin-active zones were observed as chromatographic column eluates in extracts of some batches of sap, but the nature of these could not be precisely determined. Such activity has
8
also been observed in birch spring sap and Xanthium strumarium root exudate on some occasions (Purse, 1975). There was no evidence for the presence of trans-zeatin ribotide, dihydrozeatin riboside or for ciszeatin and its derivatives in any batch of sap examined. Little is known of the origin and significance, if any, of spring sap and the solutes it contains. It has previously been shown in other woody species that levels of cytokinin activity increase in the lateral roots (Brown and Dumbroff, 1974), sap (Luckwill and Whyte, 1968;~Smid and Vardjan, 1970; Hewett and Wareing, 1973) and buds (Hewett and Wareing, 1973) just prior to or at bud-break, and the high concentrations of trans-zeatin and dihydrozeatin observed in sycamore sap in mid-February may reflect these observations. Sondheimer et al. (1971) have suggested that dihydrozeatin, together with its riboside and ribotide, are among the metabolites formed when zeatin is fed to bean axes. Apart from the report of the original isolation of this compound (Koshimizu et al., 1967), this is the only previous report of its occurrence. The difficulty in separating it from trans-zeatin suggests that it may be more widespread than reports indicate. Ribosyl-cis-zeatin residues have been demonstrated in the tRNA of all plants so far examined (see review by Hall, 1974). One method of determining whether some free cytokinins in plants arise as a result of tRNA breakdown would be to examine the extracted free cytokinins for the presence of this compound and its derivatives. Spring sap would appear to be a good source since there is less chance of cytokinins arising from tRNA of fragmented cells degraded during the extraction process. In two batches of sap in which ribosyl-trans-zeatin was identified by GC-MS, the extracts were also examined for ribosyl-cis-zeatin by bioassay, GLC and GC-MS, but none was detected. No cis-zeatin was detected in the batch of sap in which trans-zeatin was identified. While these results carry the usual conditions regarding negative evidence, the detection limits for cis-ribosylzeatin and cis-zeatin in this work were estimated to be 0.1 lag 1- 1 sap (by GLC) and 0.05 ~tg 1 t sap (by bioassay) respectively. We thank Mr. J.K. Heald for operating the GC-MS, the Science Research Council for a studentship to J.G. Purse and a grant for the purchase of the GC-MS, and the Agricultural Research Council for a fellowship to J.M. Horgan.
References Armstrong, D.J., Burrows, W.J., Evans, P.K., Skoog, F. : Isolation of cytokinins from tRNA. Biochem. Biophys. Res. Commun. 37, 451 456 (1969)
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap Babcock, D.F., Morris, R.O. : Quantitative measurement of isoprenoid nucleosides in transfer ribonucleic acid. Biochemistry 9, 3701-3705 (1970) Brown, D.C., Dumbroff, E.B. : Root growth, inhibitors, and cytokinin-like activity in sugar maple seedlings during the dormant season. Plant Physiol. Suppl. p. 71, June 1974 Dyson, W.H, Hall, R.H.: N6-(A2-isopentenyl)adenosine: Its occurrence as a free nucleoside in an autonomous strain of tobacco tissue. Plant Physiol. 50, 616~621 (1972) Halt, R.H.: Cytokinins as a probe of developmental processes. Ann. Rev. Plant Physiol. 24, 415-444 (1974) Hewett, E.W. : Cytokinins in woody plants. Ph.D. thesis. University of Wales 1973 Hewett, E.W., Wareing, P.F.: Cytokinins in Popolus X robusta: changes during chilling and bud-burst. Physiol. Plantar. (Coph) 28, 393-399 (1973) Horgan, R., Hewett, E.W., Purse, J.G., Horgan, J.M., Wareing, P.F. : Identification of a cytokinin in sycamore sap by gas chromatography-mass spectrometry. Plant Sci. Letters 1, 321-324 (1973a) Horgan, R., Hewett, E.W., Purse, J.G., Wareing, P.F.: A new cytokinin from Populus robusta. Tetrahedron Letters 30, 28272828 (1973b) Koshimizu, K., Kusaki, T., Mitsui, T., Matsubara, S.: Isolation of a cytokinin, (-)-dihydrozeatin from immature seeds of Lupinus luteus. Tetrahedron Letters 14, 1317-1320 (1967) Letham, D.S. : Zeatin, a factor inducing cell division isolated from Zea mays. Life Sciences 8, 569 573 (1963) Letham, D.S. : A new cytokinin bioassay and the naturally-occurring cytokinin complex. In: Biochemistry and physiology of plant growth substances, pp. 19 32, Wightman, F. and Setterfield, G., eds. Ottawa: Runge Press 1968 Letham, D.S.: Regulators of cell division in plant tissues XXI. Planta (Berl.) 118, 361-364 (1974) Luckwill, L.C., Whyte, P. : Hormones in the xylem sap of apple trees. In: plant growth regulators. S.C.I. Monograph No. 31, pp. 87-101, London: Society of Chemical Industry 1968 Most, B.H., Williams, J.C., Parker, K.J.: Gas chromatography of cytokinins. J. Chromatog. 38, 136-138 (1968) Parker, C.W., Letham, D.S. : Regulators of ceil division in plant tissues XVI. Planta (Berl.) 114, 199-218 Playtis, A.J., Leonard, N.J.: The synthesis of ribosyl-eis-zeatin and thin layer chromatographic separation of the cis and trans isomers of ribosyl-zeatin. Biochem. Biophys. Res. Commun. 45, l-5 (1971) Purse, J.G.: Studies on endogenous cytokinins in plants. Ph.D. thesis. University of Wales 1975 Reid, D.M., Burrows, W.J. : Cytokinin and gibberellin-like activity in the spring sap of trees. Experientia 24, 189 109 (1968) Reynolds, T.M. : Chemistry of non-enzymic browning. I. Adv. Food Res. 12, 1 52 (1963) Reynolds, T.M.: Chemistry of non-enzymic browning II. Adv. Food Res. 14, 168-285 (1965) Smid, N., Vardjan, M.: Les cytokinines dans la s6ve printani~re du bouleau Betula pendula Roth. Bioloski Vestnik 18, 27-36 (1970) Sondheimer, E., Tzou, D.S. : The metabolism of hormones during seed germination and dormancy II. Plant Physiol. 47, 516-520 (1971) Upper, C.D., Helgeson, J.P., Kemp, J.D., Schmidt, C.J.: Gasliquid chromatographic isolation of cytokinins from natural sources. Plant Physiol. 45, 543=547 (1970)' Yoshida, R., Oritani, T. : Studies on nitrogen metabolism in crop plants X. Proc. Crop Science Soc. Japan, 40, 318-324 (1971)
Received 28 November 1975; accepted 25 June 1976
Planta (Berl.) 132, 1 - 8 (1976)
9 by Springer-Verlag 1976
Cytokinins of Sycamore Spring Sap J.G. Purse*, R. Horgan, J.M. Horgan, and P.F. Wareing Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, U.K,
Summary. The cyt0kinins present in the spring sap of Acer pseudoplatanus L. were investigated. Ribosyltrans-zeatin, trans-zeatin and dihydrozeatin were isolated and identified by combined gas chromatography-mass spectrometry (GC-MS). A number of other cytokinin active fractions were observed. One of these was less polar than zeatin and did not behave as any known cytokinin. Two other fractions were more polar than ribosylzeatin and were unstable. A decomposition product of one of these was identified as ribosyl-trans-zeatin by GC-MS. The possible nature of the unstable compounds is discussed. Data on the change s in cytokinin activity of the various fractions during spring 1973 are presented and discussed.
Introduction
Most studies on cytokinins in plant tissues have yielded only limited information on the nature of the compounds present and the qualitative and quantitative changes in these compounds during plant development, due to the sole use of bioassays as the method of detection and estimation. The ability to identify all the cytokinins present in a tissue is a prerequisite for obtaining more detailed information on the action of these compounds. In certain plant tissues, cytokinins are present in sufficiently high concentrations to permit isolation of some of the compounds by crystallisation from purified extracts (e.g. Letham, 1963 ; Koshimizu et al., 1967). Approx. 70 I.tg is the minimum quantity of cytokinin that has been Present address: Milstead Laboratory of Chemical Enzymology, Broad Oak Road, Sitting, Kent, ME9 8AG, U.K. Abbreviations. GLC=gas-liquid chromatography; G C - M S = combined gas chromatography-mass spectrometry; KE=kinetin equivalents ; TLC = thin-layer chromatography; TMS = trimethylsilyl; tRNA=transfer RNA; i6Ade=6-(3-methylbut-2-enylamino)purine; i~Ado = 6-(3-methylbut-2-enylamino)-9-/?-D-ribofuranosylpurine *
obtained by this method (Parker and Letham, 1973). However, in many tissues of physiological interest cytokinins are apparently present in very low concentrations and it is usually impracticable to extract sufficient material to enable crystalline cytokinins to be obtained. We have therefore developed techniques to permit the isolation of between 1 and 10 lag of cytokinins from plant material and to identify these as their TMS derivatives by combined GC-MS. The preparation and GLC of TMS-cytokinins has been reported by Most et al. (1968) and the cytokinins of tRNA and bacterial cultures (Babcock and Morris, 1970; Upper et al., 1970) have been investigated using such procedures. However, such methods have only occasionally be used to investigate the free cytokinins of plants (Dyson and Hall, 1972; Yoshida and Oritani, 1972), mainly because of the considerable degree of prior purification required to permit successful analysis by GLC. We have used sycamore (Acer pseudoplatanus L.) spring sap as a source of cytokinins with which to develop the necessary purification procedures and we have aimed to use as mild chemical procedures as possible throughout the purification procedure. Sycamore sap has been shown to be a relatively rich source of cytokinins (Hewett, 1973). Our group has previously reported the identification of ribosyl-trans-zeatin and N6-(2-hydroxybenzyl)adenosine from sycamore sap (Horgan et al., 1973 a) and poplar leaves (Horgan et al., 1973b) respectively, using similar methods. We have also determined the changes in the amounts of cytokinins in fractions of sycamore sap obtained during spring 1973, as estimated by bioassay of the partially purified extracts. Materials and Methods Plant Material
500 1 of sycamore spring sap was collected from 15 mature, freestanding trees in the Botany Gardens, University College of Wales,
2 Aberystwyth between 23rd January and 16th April 1973, and 19th January and 14th February 1974. The sap was obtained by drilling a 25 m m diameter hole approximately 50 m m into the trunks 0.5 m above ground level, and inserting a greased cork through which was fitted a glass tube. The tube was connected to a 2.5 1 bottle containing 10 ml of toluene as bactericide. The sap was collected from these bottles daily for 6 days, after which new holes were drilled. After filtering, the sap was reduced to approximately onetenth its original volume in a cyclone evaporator at 25 ~ and further reduced to a thin syrup on a rotary evaporator, the water bath never exceeding 35~ The syrup (pH 5.5-6.0) was stored in batches in plastic bottles at - 2 0 ~ until required.
Chemicals and Solvents Analytical grade reagents were used where possible. Organic solvents were glass-distilled before use. Water used in later stages of extract purification was double-distilled in glass. Kinetin and i6Ade were obtained from Sigma Chemical Co. a n d i6Ado, zeatin, 9-/~-D-ribosylzeatin and (_+)-dihydrozeatin from Calbiochem. 6-chloro[814C]purine (10 mCi m m o l - x ) was obtained from the Radiochemical Centre, A m e r s h a m . The following cytokinins were synthesised in this laboratory by condensing 4-hydroxy-3-methylbut-trans-2enylamine with the material in brackets: trans-zeatin (6-chloropurine), 9-~-D-ribosyl-trans-zeatin (6-chloropurine-9-triacetyl-/~-Dribofuranosylpurine), trans-9-~-D-glucopyranosylzeatin (6-chloro9-tetra~acetyl-/~-D-glucopyranosyl-purine), trans-[8-14C]zeafin (6chloro[8-14C] purine).
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap in the former case and from recovered c p m in the latter. Zones with mobility similar to ribosylzeatin were eluted with E t O H : H 2 0 (4:1, v/v)for 48 h. Approximately 25% of a 5 gg sample of ribosyltrans-zeatin (estimated from G L C peak area measurements) could be recovered from a plate under these conditions. Such eluates also contained impurities arising from the plates which were detectable by G L C under the conditions used and greater proportions of water in the eluting solvent led to heavy contamination of samples.
GLC and GC-MS T M S derivativies were prepared by heating extracts or authentic c o m p o u n d s in 50 ~tl N,O-bis-trimethylsilyltrifluoracetamide:trimethylchlorosilane:hexamethyldisilazane (2:1:1, v/v) for 1 hr at 80 ~ in 0.5 ml vials. Pure N,O-bis-trimethylsilylacetamide was found to be an equally good reagent. Gas chromatography was performed on a Pye 104 fitted with dual flame ionization detectors and dual 1.65 m x 4 m m glass columns containing 2% OV-I on 100-120 mesh G a s C h r o m Q. Typical operating conditions were: carrier gas (Nz) 40 ml m i n - 1 ; H2 40 ml m i n - 1 ; air 600 ml m i n - ~ ; detectors 300 ~ oven temperature p r o g r a m m e d from 180 ~ to 300 ~ at 12 degrees m i n - 1 . Full-scale recorder deflection under these conditions was given by approximately 0.05 ~.g of TMS-cytokinin at an amplifier attenuation of 100. The G C - M S system was a Pye 104 coupled to an AEI MS30 mass spectrometer via an all-glass inlet and a silicone rubber m e m b r a n e separator. G L C conditions were the same as previously described, except that He was used as carrier gas and the mass spectrometer was used as detector. Spectra were recorded on photosensitive paper at an ionisation voltage of 24 eV.
Chromatographic Methods The following solvent systems were used: A - EtOH: HzO (92:8 v/v, containing 1 0 - 3 M HCI or CF3COOH); B - E t O H : H z O (7:13, v/v) ; C - CHC13 : M e O H (9 : 1 v/v) ; D - butan-2-ol: 12 M N H g O H (4:1 v/v). Sephadex LH-20 columns were eluted with solvents A and B. A column of LH-20 (Pharmacia, 1.8 m x 25 m m in a Chromatronix glass c o l u m n fitted with teflon bed supports) was continually run at 3 0 m l h -~ with solvent A at 2 . 1 x 1 0 4 k g m ~. The column was packed with LH-20 swollen in acetone and solvent A was forced t h r o u g h the column with a constant-flow Labotron pump. The gel expansion caused by replacement of acetone with solvent A caused a pressure increase which was maintained. Samples were injected onto the column through a septum-sealed injection port a n d up to 2 1 of eluate were collected in 50 ml or 60 ml fractions. A n o t h e r column of LH-20 (0.9 m x 25 m m , in a Chromatronix glass column fitted with teflon bed supports) was continually run at 30 ml h - 1 with solvent B. This column was packed in conventional m a n n e r and the flow was maintained continuously using a reciprocating p u m p (Metering P u m p s Ltd.). For each sample applied to the c o l u m n up to 3 1 of eluate were collected in 30 ml fractions. The recovery of [8-~4C] zeatin from both these c o l u m n s was 100% of that applied. T L C was carried out on pre-coated Merck silica gel PF2~4 plates (200 x 50 x 0.25 m m ) using solvent C (Playtis and Leonard, 1971). The plates were washed by continual development in EtOH: glacial acetic acid (4:1 v/v) for 48 h, followed by continual development with solvent C for 24 h. Extracts were c h r o m a t o g r a p h e d alongside cytokinin markers. To prevent any association of markers a n d sample the plates were scored lengthwise. Plates were continually developed until the zone thought to be of interest had migrated approx. 10 cm from the origin. Markers were located by viewing under a U V (254 nm) lamp. C h r o m a t o g r a m zones with mobility greater than or equal to trans-zeatin were eluted with 3 ml EtOH for 48 h. W h e n 5 l-tg each of i6Ado and trans-[8-~4C]zeatin were eluted under these conditions, the recoveries were 95% and 65% respectively. Recoveries were estimated from G L C peak areas
Bioassay Procedure The soybean callus assay (Miller, 1963) was used. Solutions to be assayed were placed in 100 ml conical flasks and dried on a hotplate at 40 ~ in a stream of air. 25 ml of medium was then added to the flasks, which were then autoclaved. The flasks were inoculated with three pieces of callus (approx. 0.05 g each). The cultures were grown at 26-27 ~ for three weeks under diffuse white light and the callus in each flask was then weighed. A duplicated set of kinetin controls was included in each assay. Growth of callus in an assayed fraction was expressed in gg kinetin equivalents (gg KE), this being the a m o u n t of kinetin required to elicit the same callus growth response as an active fraction in a particular assay.
Purification Procedure used on Sap Sap was thawed, adjusted to p H 3.1, filtered and percolated t h r o u g h a column of Zerolit 225 cation-exchange resin (Permutit, 52 100 mesh, 450 x 2 5 ram, N H + form, equilibrated at p H 3.1) at approx. 5 ml rain -~ (Letham, 1968). The column was washed with 1 litre of water and the effluents were combined to give fraction A. Basic substances were eluted from the column with 1 litre 1 M N H 4 O H to give fraction B. Fraction B was further purified by partitioning with organic solvents. After reduction to 500 ml the fraction was on some occasions partitioned against 500 ml EtOAc at p H 2.5. This procedure led to very slight losses of cytokinin activity from the aqueous phase (Fig. 5). The aqueous extract was partitioned against water-saturated n B u O H (4 x 500 ml) at p H 8.2. A second series of n B u O H extractions was carried out on an aqueous residue at this stage but very little cytokinin activity could be detected in it, following further purification. The n B u O H phase was reduced to dryness and dissolved in the m i n i m u m quantity of solvent D. This solution was loaded onto a cellulose column ( W h a t m a n C F l l , 240 x 20 m m , 10 g dry weight cellulose) and eluted with 350 ml solvent D, A small quan-
3
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap tity (1 gg) of trans-[8-14C]zeatin dissolved in water was taken through the procedure described so far, the ethyl acetate extraction being omitted. The overall recovery of the counts applied was 50%. The cellulose column eluate was reduced to dryness, redissolved in 2 ml solvent A and chromatographed on LH-20 in solvent A. A portion of each fraction collected was bioassayed, the remainder of each fraction being stored at - 2 0 ~ until the bioassay result was obtained. Fractions of interest were then reduced to dryness, re-dissolved in 1-2 ml solvent B and chromatographed on LH-20 in solvent B. A portion of each fraction collected was again bioassayed, the remainder being stored at - 20 ~ until required. Fractions required for analysis by G L C and GC-MS were either re-chromatographed on LH-20 in solvent B or chromatographed on silica gel in solvent C prior to silylation. All glassware used in the preparation of samples for G L C was cleaned with chromic acid. Fraction A was partitioned against n B u O H in the same manner as fraction B. The aqueous phases remaining after partitioning the two fractions were combined, reduced to 50 ml and any ribotides present were hydrolysed using alkaline phosphatase (orthophosphoric monoester phosphohydrolase [E.C.3.1.3.1 .], Sigma, calf intestinal mucosa). The solution contained 1 0 - 2 M MgC12 and incubation was for 24 h at 27 ~ p H 8. 1 ml toluene was added as bactericide. The solution was partitioned with 4 x 50 ml n B u O H and the combined organic phases were treated as the organic extract of fraction B.
Results
Identification of Ribosyl-Trans-Zeatin, Dihydrozeatin and Trans-Zeatin A 20 1 batch of sap collected 16th 17th February 1973 was purified as described in ,,Materials and Methods", the ethyl acetate extraction being omitted. Following chromatography on LH-20 in solvent A, a single peak of biological activity was observed at the elution volume of zeatin and ribosylzeatin. The remainder of this zone was chromatographed on LH-20 in solvent B, and cytokinin activity was observed in two incompletely resolved peaks at the elution volumes of ribosylzeatin and zeatin (Fig. 1. Zone A1, fraction 1718, approx. 7.5 gg K E 1-1 sap. Zone A2, fractions 20-22, approx. 50 ~tg K E 1- 1 sap). Fractions 2 3 and 24 (Fig. 1) were re-chromatographed in the same system and fractions collected were bioassayed. All biological activity eluted in Zone A2, indicating that the column had probably been over-loaded in the first run. Zones A1 and A2 were chromatographed separately on silica gel TLC plates in solvent C and zones from the plates were eluted. A portion of each eluate was bioassayed. On the plate containing zone A1, cytokinin activity was solely associated with a UV- absorbing band adjacent to the ribosyl-trans-zeatin marker. Assuming that all this band's absorbance was due to ribosylzeatin, it was estimated that approx. 8 ~tg of this compound was present. A UV-absorbing band was also observed adjacent to the ribosyl-cis-zeatin marker on
the plate, but no cytokinin activity was detected in the eluate of this zone. The remainder of each of the eluates corresponding to the two ribosylzeatin
i
AI
f
i
A2
i
q2>..
5,
ABCD IO
20
30
FRACTION NO.
Fig. 1. Cytokinin activity of fractions obtained following chromatography of a partially purified sycamore sap extract (20 1 sap, collected 16.2.73.-17.2.73.) on LH-20 in solvent B. 36 x 30 ml fractions were collected and a portion of each fraction equivalent to 1 1 of sap was assayed. Ribosylzeatin and zeatin markers elute in zones A1 and A2 respectively. Kinetin c o n t r o l s : - A , 0 ~tg 1-1; B10ggl 1;C100pgl 1;Dlmgl -I
W YZ On-
rr
sl
R W
I
0
I
I
6 12 RETENTION TIME (min) Fig. 2. G L C trace of purified sycamore sap obtained from zone A1 (Fig. 1). A portion of A1 equivalent to 14 1 of sap was chromatographed on silica gel in solvent C and the UV-absorbing band adjacent to the ribosyl-trans-zeatin marker was eluted. One-fourteenth of this eluate was bioassayed, and the remainder was dried and silylated. 2 gl aliquots of this were used for G L C analysis. Dotted lines show the result of co-injecting an aliquot of sample with 0.1 gg trans-ribosylzeatin derivatised under the same conditions. Peaks Y and Z are due to TMSs-ribosyl-trans-zeatin and TMS4-ribosyl-trans-zeatin respectively. Attenuation was 5 x 102. The TMS4-trans-ribosylzeatin peak in the extract represents approximately 0.07 gg of material injected
4
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap F I
E II
I
4"
93Ill >-
~2 /d < ~)
I
F--
]
8
s
r-R ABCD
Fig. 3. Cytokinin activity in a portion of zone A2 (Fig. 1), following chromatography on silica gel in solvent C and elution o f zones from the plate. Extract equivalent to 18 1 of sap was chromatographed and one-eighteenth of each eluate was assayed. A transzeatin marker spot migrated to zone E. O = origin, S = solvent front. Kinetincontrols:A0ggl 1;B 10/tgl-1; C 100ggl-1;D ling 1-1
la3
z
2 ce n-
I'i
Nil
123
a
0
8
RETENTION
O
b
TIME (rnin)
Fig. 4a and b. GLC traces of purified sycamore sap obtained from zone E (Fig. 3). A portion of the eluate of zone E was derivatised and aliquots of this solution were analysed by GLC. Attenuation was 20 x 102. a 3 gl aliquot of sample. Dotted line shows result of co-injecting such a sample with authentic TMS2-dihydrozeatin. b 2 •1 aliquot of sample. Dotted line shows result of co-injecting such a sample with authentic TMS2- and TMS3-trans-zeatin
isomers were silylated and a portion of each solution was gas chromatographed. In the eluate corresponding to the ribosyl-transzeatin marker a peak with the retention time of TMS4-ribosyl-trans-zeatin was observed. Bioassay of fractions of the extract collected during a preparative GLC run showed cytokinin activity only with the retention times of TMS4- and TMSs-ribosyl-transzeatin. When an aliquot of silylated extract was coinjected with authentic silylated ribosyl-trans-zeatin the peaks co-eluted (Fig. 2). Based on GLC peak area measurements, approximately 1.75 gg ribosyl-transzeatin was present in the silylated extract. Mass spec-
tra of this peak and of adjacent peaks were recorded during a GC-MS run with half the silylated material. The mass spectrum of the peak with the retention time of TMS4-ribosyl-trans-zeatin contained prominent ions at m/e 640, 639, 626, 625, 624, 551, 550, 549, 538, 537, 536, 321,320, 259, 245, 243, 230, 217, 202, 201,200, 188, 156, 147 and 103 which, by comparison with the spectrum of the authentic compound, were attributable to TMS4-ribosylzeatin. The spectrum also contained less prominent ions attributable to column bleed and preceding and succeeding peaks. All the mass spectra obtained were also examined for the presence of silylated dihydrozeatin riboside since, by analogy with dihydrozeatin and trans-zeatin, ribosyl-trans-zeatin and dihydrozeatin riboside would not be expected to separate in the chromatographic systems used prior to GLC. No evidence for its presence was found. When the eluate of the band corresponding to the ribosyl-cis-zeatin TLC marker was silylated and gas chromatographed a peak was observed with the retention time of TMS4-ribosyl-cis-zeatin, but a mass spectrum of this peak taken during a GC-MS run revealed no ions attributable to this compound. On the TLC plate containing zone A2, cytokinin activity was mainly associated with a UV-absorbing band adjacent to the trans-zeatin marker (Fig. 3, Zone E, approx. 40 Ixg KE 1 t sap) which, if entirely due to trans-zeatin was estimated to represent 1520 gg of material. A small amount of cytokinin activity of lower chromatographic mobility was also observed (Fig. 3. Zone F) and could be repeatedly demonstrated in this fraction. This zone was not further investigated, but the activity in it may have been due to a small quantity of ribosyl-trans-zeatin in zone A2. No cytokinin activity in the extract was associated with the cis-zeatin marker. The remainder of the eluate of zone C was silylated and a 2 lal portion was gas chromatographed. The only peaks observed were shown to co-elute with TMS/-dihydrozeatin, TMS3-zeatin and TMS2-zeatin when further portions of extract were co-injected with authentic compounds (Fig. 4). Bioassay of fractions collected during a preparative GLC run with a further portion of extract showed cytokinin activity to be associated with this group of peaks only. Mass spectral scans recorded during two GC-MS runs with further portions of the extract confirmed the presence of these compounds. The mass spectrum of the peak with the retention time of TMS2-dihydrozeatin contained ions at m/e 365, 351, 350, 292, 276, 275, 264, 262, 260, 235, 234, 221,220, 207, 192, 162, 148 and 135, indicative of the presence of this compound. The mass spectrum on the shoulder of the above peak, co-eluting with TMS3-trans-zeatin, contained ions at
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap AI
A2
5
A3
2"
G H
~-.-4H
I-5-
I-O
125
u)
0.5 84
I J
5
~b >.-
0 6
ABC
u 4
2-
O-
2b FRACTION
3b
ABCD b
NO.
Fig. Sa and b. Cytokinin activity in fractions obtained following chromatography of a ethyl acetate extract and b n-butanol extract of partially purified sycamore sap (30 1, collected 1st Feb.-3rd Feb. 1973) on LH-20 in solvent B. 3 0 x 3 0 ml fractions were collected and the equivalent of 1 1 of sap from each fraction was assayed. Zones A1, A2 and A3 correspond to the elution positions of ribosylzeatin, zeatin and i6Ado respectively. Kinetin controls: A0ggl 1;BlOp.gl-1;ClOOiagl-l;Dlmgl 1
m/e 436, 435, 434, 422, 421, 420, 348, 347, 346, 345, 332, 306, 305, 304 and 272, attributable to TMS3zeatin, together with ions corresponding to TMSzdihydrozeatin. The mass spectrum of the peak with the retention time of TMS2-trans-zeatin contained ions at m/e 363, 349, 348, 274, 273, 272, 261, 260, 258, 232, 201, 160 and 146, indicating the presence of this compound, together with other ions attributable to TMSa-dihydrozeatin. The amounts of transzeatin and dihydrozeatin thus isolated and identified were estimated by GLC peak size to represent 0.3 gg 1- ~ sap and 0.5 pg 1- 1 sap respectively. These figures take no account of losses of these compounds incurred during the purification procedure. No cytokinin activity was detected in any sap which was examined for the possible presence of cytokinin ribotides as described in "Materials and Methods". Investigation of a Further Cytokinin-active Fraction Present in two Batches of Sap A zone of cytokinin activity (zone A3 on the LH-20 column eluted with solvent B) was observed in two batches of sap collected lst-3rd February 1973 (batch I) and 6th-9th February 1973 (batch 2). It could not be detected in other batches collected in 1973-74 in spite of considerable investigation. Both
Fig. 6. Cytokinin activity in a portion of zone A3 (Fig. 5 b, equivalent to three litres of sap), following chromatography on silica gel in solvent C and elution of zones from the plate, Bars G and H indicate positions of i6Ado and i6Ade markers respectively. O=origin, S=solvent front. Kinetin controls: A 0 pg 1 1; B 10 pg 1 1; C 1 0 0 g g l - 1
batches were purified according to the standard procedure, the ethyl acetate extraction being included for batch 1. The ethyl acetate and butanol extracts of this batch were chromatographed separately on LH-20 in solvent B and a portion of each fraction collected was bioassayed (Fig. 5). Very little of the cytokinin activity present in the extract passed into the organic phase during the partitioning with ethyl acetate. However, slight callus growth was observed in zone A3 of this fraction and cytokinin activity could consistently be demonstrated in it throughout its subsequent purification. A portion of zone A3 obtained from the butanol fraction was chromatographed on a silica gel TLC plate in solvent C alongside i6Ade and i6Ado markers. Ten zones from the plate were eluted and a portion of each eluate was bioassayed. The only zone of biological activity observed was more mobile than both markers and co-incided with the only UV-absorbing spot in the extract (Fig. 6). Further details of the isolation and identification of this compound will be presented in a separate paper. Unstable Fractions Observed in Extracts In some batches of sap which were subjected to the normal purification procedure, cytokinin activity was observed in fractions collected from the LH-20 column eluted with solvent B at and before the elution volume of 9-glucosylzeatin (zones A4 and A5 respectively). In some such batches these fractions contained almost all the cytokinin activity in the extract, while in others they represented only part of the total cytokinin activity. When these fractions were re-chromatographed in the same system, cytokinin activity was usually observed at the elution volumes of ribosylzea-
6
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap A5 I
A4 II
AI I
~
A2 i
I
..... I
m 3-5.:]. .j 2 l < u
IS]
ABC b
,8
2b
FRACTION
NO.
Fig. 7. a Cytokinin activity in an extract of sycamore sap (45 1, collected 30th Jan.-3rd Feb. 1974), folIowing chromatography on LH-20 in solvent B. 24 x 30 ml fractions were collected and a portion of each fraction equivalent to 1 1 of sap was assayed, b The remainder of each fraction from a was stored for four weeks at - 2 0 ~ and fractions 10-12 were then re-chromatographed in the same system. 28 x 30 ml fractions were collected and portions of each fraction equivalent to 1 1 of sap were bioassayed. 9-glucosylzeatin, ribosylzeatin and zeatin elute in zones A4, A1 and A2 respectively. Kinetin controls A 0 lag 1- ~ ; B 10 lag 1 ~ ; C 100 lag 1-1
Ld U3 Z Iii rY
kW rm
J
O
12 RETENTION
TIME (rnin)
Fig. 8. G L C trace of silylated material obtained from partially purified sycamore sap fractions containing the active decomposition product of an unstable cytokinin-active fraction (Fig. 7 b, fractions 19-20). The dried material was silylated and a 2 lal aliquot was injected into the gas chromatograph. The dashed line indicates the result of co-injecting a further 2 gl aliquot with 0.02 lag trans-ribosylzeatin derivatised in the same manner. Attenuation 2 x 102
tin and/or zeatin (Fig. 7), although in some cases all or part of the cytokinin activity revealed in the earliereluting zone. A batch of sap (45 1, collected 30th January-3rd February 1974) was purified in the usual manner,
the ethyl acetate extraction being omitted. Following chromatography on LH-20 in solvent B, cytokinin activity with an elution volume of 270-420 ml was observed (Fig. 7a). Following storage in solvent B at - 2 0 ~ for 4 weeks, fractions 10-12 (elution volume 270-360 ml) were re-chromatographed on LH-20 in solvent B, portions of each fraction collected being bioassayed. Cytokinin activity was observed mainly at the elution volume of ribosylzeatin (Fig. 7b, fractions 19 20). The remainder of fractions 19 and 20 were silylated. Gas chromatography of a portion of the silylated fractions gave a peak which co-chromatographed with authentic TMS4-ribosyl-trans-zeatin (Fig. 8), and the presence of this compound was confirmed with a mass spectral scan of this peak taken during a GC-MS run with the remainder of the sampie. The mass spectrum obtained contained prominent ions at m/e 639, 624, 550, 549, 536, 320, 276, 274, 260, 259, 245, 243, 230, 217, 204, 201, 200, 188, 156, 147 and 103 at very similar relative intensities to the authentic compound. Thus the principal cytokinin active decomposition product obtained from the unstable fraction was identified as ribosyl-trans-zeatin. The similar behaviour of the other unstable fractions observed suggested that they all decomposed to give either zeatin or ribosylzeatin. The order of elution of cytokinins from the LH-20 column eluted with solvent B closely parallel the partition coefficients of cytokinins between nBuOH and aqueous solutions at pH 7 or 8 (Letham, 1974; Purse, 1975) when the partition coefficients are arranged in increasing order, suggesting that this column operated mainly be reverse-phase partition rather than by adsorption as has previously been stated (Armstrong etal., 1969). Thus, in general, polar compounds would be expected to elute more rapidly from this column than less polar ones. Thus the compounds in the unstable active fractions were probably more polar than their active decomposition products. It is possible that these early-eluting fractions contained naturally-occurring, polar cytokinins or else were artefacts of the sap collection and/or purification procedure. In the latter case, such artefacts could be due to the formation of compounds more polar than ribosylzeatin or could be due to an association of a physical nature, e.g. association with another compound during chromatography. While insufficient evidence was available to distinguish between these possibilities, the evidence suggested that they were artefacts of some kind, since there is no obvious reason why unstable compounds should survive all the purification procedure prior to chromatography on LH-20 and yet decompose in aqueous ethanol at - 20 ~ Also, when a second series of butanol extraction was performed on the basic etuate from the ca-
J.G. Purse et al. : Cytokininsof SycamoreSpring Sap tion-exchange column, the extract contained very little cytokinin activity, even though if polar cytokinins had been present they would have incompletely partitioned into butanol during the first series of extractions, so that the butanol from the second series of extractions would also contain such cytokinins. A type of reaction which could lead to polar derivatives of zeatin and ribosylzeatin is reaction of a primary or secondary amine with a reducing sugar to yield an aldosamine or ketosamine (Reynolds, 1963, 1965). This type of reaction is said to occur in many syrups and freeze-dried foods and proceeds under mild conditions. The products formed are readily hydrolysed and always yield the parent amine. Attempts were made to carry out such a reaction by (a) adding 5 gg of authentic ribosyl-trans-zeatin to 1 1 of freshly collected sycamore sap, taking to dryness in vacuo and extracting the cytokinins by cation-exchange, butanol partition and chromatography on LH-20 in solvent B, (b) warming trans-[8-14C]zeatin with glucose or fructose under conditions similar to those used during the purification procedure, immediately followed by chromatography on LH-20 in solvent B. In neither case was any active material detected at elution volumes other than those of ribosylzeatin and zeatin respectively. Thus the nature of the cytokinin activity observed in these fractions remains to be elucidated.
Changes in Cytok&in Content of Sap Collected in Spring 1973 Figure 9 shows the changes in cytokinin activity observed in zones A1, A2, A 4 + A 5 and A1 + A 4 + A 5 during spring 1973. All points on these graphs are estimates of the total biological activity in the appropriate zone(s) from the LH-20 column eluted with solvent B, following assay of extract equivalent to 1 1 of sap. Sap was collected either on the indicated date or over a period of several days, in which case the date in the middle of this period is shown on the graphs. Sap was extracted according to the complete extraction procedure, except when only 1 2 1 of sap was being processed, when it was purified by cation-exchange , butanol partition and chromatography on LH-20 in solvent B only. Bud swelling on the trees was apparent in early March and budburst occurred around 20th March. No activity was detectable in zone A2 from sap collected in late January, but the activity in this zone reached a maximum in mid-February and subsequently declined somewhat. Both trans-zeatin and dihydrozeatin were identified in the sap at the time when it contained maximum activity, and dihydrozeatin was present in greater amounts at this time. However,
7
o 12 __
A2
oJ S t ~l~176
A,
1
O j
i
JAN.
i
FEB.
i
MAR.
APR,
Fig. 9. Changes in cytokinin activity in zones A2, AI, A4+A5 and A1 +A4+A5 of sycamore sap extracts during spring 1973, estimated from the cytokinin activity in fractions obtained from extracts chromatographedon LH-20 in solvent B since dihydrozeatin possesses only 10-20% of the cytokinin activity of trans-zeatin in the soybean assay, most of the activity shown at this point on the plot is presumably due to trans-zeatin. For reasons discussed above, it is probable that zones A4 and A5 are artefacts derived mainly from ribosylzeatin and probably zeatin also or are unstable, naturally-occurring derivatives of these compounds. Thus the principal active compound detected in bioassays of these zones may be ribosylzeatin itself. While the cytokinin activity detected in zones A1 and A 4 + A 5 fluctuated considerably during spring 1973, the total activity in these zones remained remarkably constant, apart from a peak in early February. Discussion
Previous work has indicated the presence of cytokinins in the spring sap of Acer pseudoplatanus (Reid and Burrows, 1968; Hewett, 1973) and our group has already reported the identification of ribosyltrans-zeatin from this source by GC-MS. The purification techniques have now been refined sufficiently to permit the separation, isolation and identification of further cytokinins. In the present work, the isolation and identification of ribosyl-trans-zeatin, transzeatin and dihydrozeatin using such techniques are described. Details of the characterisation of the cytokinin-active compound in zone A3 will be published separately. Unstable, cytokinin-active zones were observed as chromatographic column eluates in extracts of some batches of sap, but the nature of these could not be precisely determined. Such activity has
8
also been observed in birch spring sap and Xanthium strumarium root exudate on some occasions (Purse, 1975). There was no evidence for the presence of trans-zeatin ribotide, dihydrozeatin riboside or for ciszeatin and its derivatives in any batch of sap examined. Little is known of the origin and significance, if any, of spring sap and the solutes it contains. It has previously been shown in other woody species that levels of cytokinin activity increase in the lateral roots (Brown and Dumbroff, 1974), sap (Luckwill and Whyte, 1968;~Smid and Vardjan, 1970; Hewett and Wareing, 1973) and buds (Hewett and Wareing, 1973) just prior to or at bud-break, and the high concentrations of trans-zeatin and dihydrozeatin observed in sycamore sap in mid-February may reflect these observations. Sondheimer et al. (1971) have suggested that dihydrozeatin, together with its riboside and ribotide, are among the metabolites formed when zeatin is fed to bean axes. Apart from the report of the original isolation of this compound (Koshimizu et al., 1967), this is the only previous report of its occurrence. The difficulty in separating it from trans-zeatin suggests that it may be more widespread than reports indicate. Ribosyl-cis-zeatin residues have been demonstrated in the tRNA of all plants so far examined (see review by Hall, 1974). One method of determining whether some free cytokinins in plants arise as a result of tRNA breakdown would be to examine the extracted free cytokinins for the presence of this compound and its derivatives. Spring sap would appear to be a good source since there is less chance of cytokinins arising from tRNA of fragmented cells degraded during the extraction process. In two batches of sap in which ribosyl-trans-zeatin was identified by GC-MS, the extracts were also examined for ribosyl-cis-zeatin by bioassay, GLC and GC-MS, but none was detected. No cis-zeatin was detected in the batch of sap in which trans-zeatin was identified. While these results carry the usual conditions regarding negative evidence, the detection limits for cis-ribosylzeatin and cis-zeatin in this work were estimated to be 0.1 lag 1- 1 sap (by GLC) and 0.05 ~tg 1 t sap (by bioassay) respectively. We thank Mr. J.K. Heald for operating the GC-MS, the Science Research Council for a studentship to J.G. Purse and a grant for the purchase of the GC-MS, and the Agricultural Research Council for a fellowship to J.M. Horgan.
References Armstrong, D.J., Burrows, W.J., Evans, P.K., Skoog, F. : Isolation of cytokinins from tRNA. Biochem. Biophys. Res. Commun. 37, 451 456 (1969)
J.G. Purse et al. : Cytokinins of Sycamore Spring Sap Babcock, D.F., Morris, R.O. : Quantitative measurement of isoprenoid nucleosides in transfer ribonucleic acid. Biochemistry 9, 3701-3705 (1970) Brown, D.C., Dumbroff, E.B. : Root growth, inhibitors, and cytokinin-like activity in sugar maple seedlings during the dormant season. Plant Physiol. Suppl. p. 71, June 1974 Dyson, W.H, Hall, R.H.: N6-(A2-isopentenyl)adenosine: Its occurrence as a free nucleoside in an autonomous strain of tobacco tissue. Plant Physiol. 50, 616~621 (1972) Halt, R.H.: Cytokinins as a probe of developmental processes. Ann. Rev. Plant Physiol. 24, 415-444 (1974) Hewett, E.W. : Cytokinins in woody plants. Ph.D. thesis. University of Wales 1973 Hewett, E.W., Wareing, P.F.: Cytokinins in Popolus X robusta: changes during chilling and bud-burst. Physiol. Plantar. (Coph) 28, 393-399 (1973) Horgan, R., Hewett, E.W., Purse, J.G., Horgan, J.M., Wareing, P.F. : Identification of a cytokinin in sycamore sap by gas chromatography-mass spectrometry. Plant Sci. Letters 1, 321-324 (1973a) Horgan, R., Hewett, E.W., Purse, J.G., Wareing, P.F.: A new cytokinin from Populus robusta. Tetrahedron Letters 30, 28272828 (1973b) Koshimizu, K., Kusaki, T., Mitsui, T., Matsubara, S.: Isolation of a cytokinin, (-)-dihydrozeatin from immature seeds of Lupinus luteus. Tetrahedron Letters 14, 1317-1320 (1967) Letham, D.S. : Zeatin, a factor inducing cell division isolated from Zea mays. Life Sciences 8, 569 573 (1963) Letham, D.S. : A new cytokinin bioassay and the naturally-occurring cytokinin complex. In: Biochemistry and physiology of plant growth substances, pp. 19 32, Wightman, F. and Setterfield, G., eds. Ottawa: Runge Press 1968 Letham, D.S.: Regulators of cell division in plant tissues XXI. Planta (Berl.) 118, 361-364 (1974) Luckwill, L.C., Whyte, P. : Hormones in the xylem sap of apple trees. In: plant growth regulators. S.C.I. Monograph No. 31, pp. 87-101, London: Society of Chemical Industry 1968 Most, B.H., Williams, J.C., Parker, K.J.: Gas chromatography of cytokinins. J. Chromatog. 38, 136-138 (1968) Parker, C.W., Letham, D.S. : Regulators of ceil division in plant tissues XVI. Planta (Berl.) 114, 199-218 Playtis, A.J., Leonard, N.J.: The synthesis of ribosyl-eis-zeatin and thin layer chromatographic separation of the cis and trans isomers of ribosyl-zeatin. Biochem. Biophys. Res. Commun. 45, l-5 (1971) Purse, J.G.: Studies on endogenous cytokinins in plants. Ph.D. thesis. University of Wales 1975 Reid, D.M., Burrows, W.J. : Cytokinin and gibberellin-like activity in the spring sap of trees. Experientia 24, 189 109 (1968) Reynolds, T.M. : Chemistry of non-enzymic browning. I. Adv. Food Res. 12, 1 52 (1963) Reynolds, T.M.: Chemistry of non-enzymic browning II. Adv. Food Res. 14, 168-285 (1965) Smid, N., Vardjan, M.: Les cytokinines dans la s6ve printani~re du bouleau Betula pendula Roth. Bioloski Vestnik 18, 27-36 (1970) Sondheimer, E., Tzou, D.S. : The metabolism of hormones during seed germination and dormancy II. Plant Physiol. 47, 516-520 (1971) Upper, C.D., Helgeson, J.P., Kemp, J.D., Schmidt, C.J.: Gasliquid chromatographic isolation of cytokinins from natural sources. Plant Physiol. 45, 543=547 (1970)' Yoshida, R., Oritani, T. : Studies on nitrogen metabolism in crop plants X. Proc. Crop Science Soc. Japan, 40, 318-324 (1971)
Received 28 November 1975; accepted 25 June 1976
