Planta 135, 2 8 5 - 288(1977)
P
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9 by Springer-Verlag 1977
A Cytokinin Glueoside from the Leaves of Phaseolus vulgaris L. T.L. W a n g * , A . G . Thompson**, and R. Horgan Department of Botany and Microbiology, The University College of Wales, Penglais, Aberystwyth SY23 3DA, U.K.
Phaseolus vulgaris plants decapitated above the primary leaves accumulate high cytokinin activity. The major cytokinin in these leaves was identified by Sephadex LH 20 chromatography, sensitivity to/%glucosidase and permanganate oxidation, and by combined gas chromatography-mass spectrometry as 6-(4O-/%D-glucosyl-3-methylbutylamino) purine, (dihydrozeatin-O-/%D-glucoside). A possible reason for the persistance of this compound in the primary leaves is discussed.
Abstract.
Key words: C y t o k i n i n - Leaves - Phaseolus.
plants as 6-(4-O-fi-D-glucosyl-3-methylbutylamino) purine (dihydrozeatin-O-fi-D-glucoside).
Materials and Methods Plant Material Plants ofPhaseolus vulgaris L. var ~ Canadian Wonder" were grown in a greenhouse in John Innes No. 2 compost. They were maintained at 20~ and under long days (18 h). Once the first trifoliate leaves began to expand, the plants were decapitated above the primary leaves and the axillary buds removed. Any new axillary shoot growth was removed immediately as it appeared. After 50 days the primary leaves were harvested and frozen in liquid air.
Introduction Chromatography-Paper
Roots have long been considered to be a primary source of cytokinins in higher plants (Skene, 1975) although most of the evidence for this has been based on indirect surgical experiments. The observation of Engelbrecht (1972) that high levels of cytokinin accumulated in the laminae of isolated bean leaves when roots developed on the petiole, represents one of the most spectacular demonstrations of cytokinin production by roots. The large build up of cytokinin suggested that this system would be a very suitable one for the study of cytokinin production by the roots using more direct methods. It has been shown that decapitated bean plants accumulate cytokinin in a similar manner to the rooted bean leaves of Engelbrecht (Wang, 1977) and therefore, as a prelude to biosynthesis studies, we have identified the major cytokinin in decapitated bean * Present address." Botany Department, University of Leicester, Leicester LEI 7RH, U.K. ** Present address." The Polytechnic, Holly Bank Road, Huddersfield HD3 3BP, U.K. Abbreviations." BSA=Bis-(trimethylsilyl)-acetamide; DHZ=dihydrozeatin; DHZOG=dihydrozeatin-O-fi-D-glucoside; TMS=trimethylsilyl; Z = zeatin
Whatman No. 3MM chromatography paper pre-washed successively in water, ethanol and Solvent C was used. Chromatograms run in Solvent C were developed descendingly for approximately 30 cm and dried for 24 h. Chromatograms run in Solvents D and E were developed ascendingly for 15 cm. Where appropriate 2 mm strips from the edge of the chromatograms were used for bioassay.
Column 2.5 x (70-80) cm Sephadex LH 20 columns were used. They were eluted at a flow rate of 30 cm 3 h - 1 and 30 cm 3 fractions collected. 0.1 cm 3 samples were withdrawn for bioassay.
Thin-layer TLC was carried out using Merck 5 x 20 cm, 0.25 mm thick silicagel 60 PF254 pre-coated plates using Solvent F. Material was removed from the layers by extracting with 80% ethanol (v/v).
Gas-liquid Chromatography ( GLC) and Combined Gas Chromatography-mass Spectrometry (GCMS) Samples for chromatography were treated with fi-glucosidase as described below. TMS derivatives were made by reacting 20 gl BSA
286 with the sample in a sealed tube at 90 ~C for 1 h. GLC was carried out on a Pye 104 gas chromatography fitted with dual flame ionisation detectors. Glass columns (4 mm x 1.65 m) were packed with 2% OV-I on 100-120mesh Gas Chrom Q. Nitrogen carrier gas flow rate was 40 cm 3 rain- 1. A temperature program from 180300 ~C at 12~ rain- 1 was employed. Preparative GLC was performed using a modified Pye 104 gas chromatograph with a split ratio of about 1 : 4 (detector: trap). GCMS was carried out on a AEI MS 30 mass spectrometer linked to a Pye 104 GC via a silicon rubber membrane separator. Spectra were recorded at 3 s per mass decade using a DS 50 data system.
T.L. Wang et al.:
A Cytokinin Glucoside from Leaves
Permanganate Oxidation Oxidation of the samples by permanganate was carried out after the method of Sondheimer and Tzou (1971). Once treated the samples were chromatographed on paper in Solvent E.
Bioassays The assays used were either the tobacco stem pith callus assay (Murashige and Skoog, 1972) or the soybean cotyledonary callus assay (Miller, 1968).
Solvents A - 35 % ethanol (v/v) ; B -- water; C-- sec-butanol : 25 % ammonia (4 : 1, v/v) ; D - n-butanol:acetic acid:water (12:3 : 5, v/v) ; E - 35 % ethanol (v/v); F - n - b u t a n o l : 14 M ammonia:water (6:1:2. v/v, upper phase).
Cytokinin Extraction 1200 g of bean leaves were homogenised in a Wareing blendor in 10 dm 3 of 80% methanol (v/v) and stirred overnight at 4~ The mixture was filtered, the residue re-extracted in a further 10 dm 3 of 80% methanol, and stirred overnight. After filtering again, the combined filtrates were reduced to dryness in vacuo at 30 ~C. The dry residue was suspended in about 200 cm 3 of distilled water, adjusted to pH 3 and frozen. After thawing, the suspension was centrifuged at 15,000 x g for 1 h to remove lipid material. The supernatant was passed through a Zerolit 225 (NH~ form) cation exchange column (1000 cm 3 bed volume) equilibrated to pH 3.1. The column was washed with 5 column volumes of water (pH 3.1), and then eluted with 5 column volumes of 2 mol dm 3 ammonia solution. The ammonia eluate was reduced in vacuo to approximately 250 cm 3, adjusted to pH 8 and partitioned 5 times against equal volumes of water-saturated n-butanol. The combined butanol phases were reduced to dryness and dissolved in 2.5 cm 3 Solvent A. This was applied to a Sephadex LH 20 column eluted with Solvent A. The active fractions (11 and 12, see Fig. la) were taken to dryness and dissolved in 1 cm 3 water. This sample was chromatographed on a Sephadex LH 20 column eluted with Solvent B. The active fractions from this column (20 and 21, see Fig. 1 b) were reduced to dryness, dissolved in 0.25 cm 3 Solvent E and subjected to paper chromatography in Solvent C. The biologically active region (Rf0.21-0.32), was eluted with Solvent E. The eluate was taken to dryness and chromatographed as a spot in Solvent D. A single UV absorbing spot was observed at Rf0.5. This spot was eluted with Solvent E, the solvent evaporated to dryness and the sample used for further analysis by GCMS and UV spectroscopy.
Results A reassessment of Engelbrecht's (1972) results on the accumulation of cytokinin in the laminae of rooted bean leaves has confirmed her observations that the cytokinin produced by this system was a hitherto unidentified compound. To obtain sufficient of this compound for structure determination, we have used deca-
a
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2.584
10' 300
20 600
b
Z9G i
30 F No 900 E Vol
'~ 10 KINETIN
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u.. Q. 2.5
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Treatment with [3-Glucosidase Samples for treatment were dissolved in Solvent E and dried into small vials. They were then incubated with 25 lag of/~-glucosidase (Boehringer Corp. Ltd.) in 0.1 mol dm -3 sodium acetate buffer, pH 5.3, overnight at 37 ~ C. After drying, samples were removed from the vial using 10 lal of ethanol followed by 10 lal 80% ethanol (v/v).
Glucose Detection Samples for glucose assay were treated with /~-glucosidase as described. The residue was dissolved in 25 gl Solvent E and chromatographed in Solvent B on paper. The glucose was visualised using " G l u c o s t a t " reagent (Sigma Chemical Co. Ltd.).
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Fig. 1. a Tobacco callus bioassay of bean leaf extract chromatographed on Sephadex LH 20. A 2.5 x 75 column was eluted with Solvent A at a flow rate of 30 cm 3 h - 1 30 cm 3 fractions were collected and 0.1 cm 3 (equivalent to about 4 g fresh weight of tissue) withdrawn for assay. Fractions 11 and 12 were combined and used for further purification, b Tobacco callus assay of bean leaf extract chromatographed on Sephadex LH 20. A 2.5 x 72 cm column was eluted with Solvent B at a flow rate of 30 c m ? h 1.30 cm 3 fractions were collected and 0.1 cm 3 withdrawn for assay. Fractions 20 and 21 were combined and used for paper chromatography
T.L. Wang et al.: A Cytokinin Glucoside from Leaves
287
KINETIN~gl I
and similarly chromatographed. The results indicated that the unknown cytokinin was degraded by/%glucosidase to a biologically active compound or compounds which chromatographed with zeatin in Solvent F. Thus it was concluded that the unknown cytokinin was probably a /LD-glucoside. Permanganate treatment of further aliquots, after enzymic removal of the glucose moiety and chromatography in Solvent D followed by bioassay, showed that the aglycone was resistant to such oxidation. Hence the compound did not possess an isopentenyl side chain. Final purification of the compound was achieved by paper chromatography in Solvents C and F. An ultraviolet spectrum of the purified compound in Solvent E gave a •max at 269 nm while, at high pH (ammoniacal Solvent E), a shift to 275 nm with a shoulder at 284 nm was observed. Such a spectrum is characteristic of N6-substituted purines with no other ring substituents. From the absorption obtained approximately 40 ~tg kg- 1 of the purified compound was isolated (based on an em,xof 14.6 x 1 0 3 for zeatin). The purified compound was hydrolysed with /% glucosidase and the glucose released positively identified using glucose oxidase (Glucostat). The aglycone was subjected to GCMS and preparative GLC as the TMS derivative. GCMS of the aglycone gave the following ions at m/e ratios of: M § 365 (12.1), 350 (19.3), 276 (3.4), 275 (4.1), 274 (2.3), 262 (9.6), 260 (5.5), 236 (6.5), 235 (32.4), 234 (100), 233 (4.8), 232 (5.0), 222 (6.5), 221 (25.5), 220 (73.4), 193 (4.7), 192 (12.1), 162 (5.7) and it was therefore identified as dihydrozeatin. Preparative GLC followed by bioassay showed that all the cytokinin activity in the sample was confined to the dihydrozeatin-TMS peak (Fig. 2). On the basis of the above results, the major cytokinin in the leaves of decapitated bean plants was tentatively identified as 6-(4-O-fl-D-glucosyl-3-methylbutylamino) purine (dihydrozeatin-O-/~-D-glucoside). The identification of this compound, although not confirmed by synthesis, represents the most positive proof to date of the occurrence of endogenous cytokinin glucosides in higher plants.
2.50-
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P
l
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9 by Springer-Verlag 1977
A Cytokinin Glueoside from the Leaves of Phaseolus vulgaris L. T.L. W a n g * , A . G . Thompson**, and R. Horgan Department of Botany and Microbiology, The University College of Wales, Penglais, Aberystwyth SY23 3DA, U.K.
Phaseolus vulgaris plants decapitated above the primary leaves accumulate high cytokinin activity. The major cytokinin in these leaves was identified by Sephadex LH 20 chromatography, sensitivity to/%glucosidase and permanganate oxidation, and by combined gas chromatography-mass spectrometry as 6-(4O-/%D-glucosyl-3-methylbutylamino) purine, (dihydrozeatin-O-/%D-glucoside). A possible reason for the persistance of this compound in the primary leaves is discussed.
Abstract.
Key words: C y t o k i n i n - Leaves - Phaseolus.
plants as 6-(4-O-fi-D-glucosyl-3-methylbutylamino) purine (dihydrozeatin-O-fi-D-glucoside).
Materials and Methods Plant Material Plants ofPhaseolus vulgaris L. var ~ Canadian Wonder" were grown in a greenhouse in John Innes No. 2 compost. They were maintained at 20~ and under long days (18 h). Once the first trifoliate leaves began to expand, the plants were decapitated above the primary leaves and the axillary buds removed. Any new axillary shoot growth was removed immediately as it appeared. After 50 days the primary leaves were harvested and frozen in liquid air.
Introduction Chromatography-Paper
Roots have long been considered to be a primary source of cytokinins in higher plants (Skene, 1975) although most of the evidence for this has been based on indirect surgical experiments. The observation of Engelbrecht (1972) that high levels of cytokinin accumulated in the laminae of isolated bean leaves when roots developed on the petiole, represents one of the most spectacular demonstrations of cytokinin production by roots. The large build up of cytokinin suggested that this system would be a very suitable one for the study of cytokinin production by the roots using more direct methods. It has been shown that decapitated bean plants accumulate cytokinin in a similar manner to the rooted bean leaves of Engelbrecht (Wang, 1977) and therefore, as a prelude to biosynthesis studies, we have identified the major cytokinin in decapitated bean * Present address." Botany Department, University of Leicester, Leicester LEI 7RH, U.K. ** Present address." The Polytechnic, Holly Bank Road, Huddersfield HD3 3BP, U.K. Abbreviations." BSA=Bis-(trimethylsilyl)-acetamide; DHZ=dihydrozeatin; DHZOG=dihydrozeatin-O-fi-D-glucoside; TMS=trimethylsilyl; Z = zeatin
Whatman No. 3MM chromatography paper pre-washed successively in water, ethanol and Solvent C was used. Chromatograms run in Solvent C were developed descendingly for approximately 30 cm and dried for 24 h. Chromatograms run in Solvents D and E were developed ascendingly for 15 cm. Where appropriate 2 mm strips from the edge of the chromatograms were used for bioassay.
Column 2.5 x (70-80) cm Sephadex LH 20 columns were used. They were eluted at a flow rate of 30 cm 3 h - 1 and 30 cm 3 fractions collected. 0.1 cm 3 samples were withdrawn for bioassay.
Thin-layer TLC was carried out using Merck 5 x 20 cm, 0.25 mm thick silicagel 60 PF254 pre-coated plates using Solvent F. Material was removed from the layers by extracting with 80% ethanol (v/v).
Gas-liquid Chromatography ( GLC) and Combined Gas Chromatography-mass Spectrometry (GCMS) Samples for chromatography were treated with fi-glucosidase as described below. TMS derivatives were made by reacting 20 gl BSA
286 with the sample in a sealed tube at 90 ~C for 1 h. GLC was carried out on a Pye 104 gas chromatography fitted with dual flame ionisation detectors. Glass columns (4 mm x 1.65 m) were packed with 2% OV-I on 100-120mesh Gas Chrom Q. Nitrogen carrier gas flow rate was 40 cm 3 rain- 1. A temperature program from 180300 ~C at 12~ rain- 1 was employed. Preparative GLC was performed using a modified Pye 104 gas chromatograph with a split ratio of about 1 : 4 (detector: trap). GCMS was carried out on a AEI MS 30 mass spectrometer linked to a Pye 104 GC via a silicon rubber membrane separator. Spectra were recorded at 3 s per mass decade using a DS 50 data system.
T.L. Wang et al.:
A Cytokinin Glucoside from Leaves
Permanganate Oxidation Oxidation of the samples by permanganate was carried out after the method of Sondheimer and Tzou (1971). Once treated the samples were chromatographed on paper in Solvent E.
Bioassays The assays used were either the tobacco stem pith callus assay (Murashige and Skoog, 1972) or the soybean cotyledonary callus assay (Miller, 1968).
Solvents A - 35 % ethanol (v/v) ; B -- water; C-- sec-butanol : 25 % ammonia (4 : 1, v/v) ; D - n-butanol:acetic acid:water (12:3 : 5, v/v) ; E - 35 % ethanol (v/v); F - n - b u t a n o l : 14 M ammonia:water (6:1:2. v/v, upper phase).
Cytokinin Extraction 1200 g of bean leaves were homogenised in a Wareing blendor in 10 dm 3 of 80% methanol (v/v) and stirred overnight at 4~ The mixture was filtered, the residue re-extracted in a further 10 dm 3 of 80% methanol, and stirred overnight. After filtering again, the combined filtrates were reduced to dryness in vacuo at 30 ~C. The dry residue was suspended in about 200 cm 3 of distilled water, adjusted to pH 3 and frozen. After thawing, the suspension was centrifuged at 15,000 x g for 1 h to remove lipid material. The supernatant was passed through a Zerolit 225 (NH~ form) cation exchange column (1000 cm 3 bed volume) equilibrated to pH 3.1. The column was washed with 5 column volumes of water (pH 3.1), and then eluted with 5 column volumes of 2 mol dm 3 ammonia solution. The ammonia eluate was reduced in vacuo to approximately 250 cm 3, adjusted to pH 8 and partitioned 5 times against equal volumes of water-saturated n-butanol. The combined butanol phases were reduced to dryness and dissolved in 2.5 cm 3 Solvent A. This was applied to a Sephadex LH 20 column eluted with Solvent A. The active fractions (11 and 12, see Fig. la) were taken to dryness and dissolved in 1 cm 3 water. This sample was chromatographed on a Sephadex LH 20 column eluted with Solvent B. The active fractions from this column (20 and 21, see Fig. 1 b) were reduced to dryness, dissolved in 0.25 cm 3 Solvent E and subjected to paper chromatography in Solvent C. The biologically active region (Rf0.21-0.32), was eluted with Solvent E. The eluate was taken to dryness and chromatographed as a spot in Solvent D. A single UV absorbing spot was observed at Rf0.5. This spot was eluted with Solvent E, the solvent evaporated to dryness and the sample used for further analysis by GCMS and UV spectroscopy.
Results A reassessment of Engelbrecht's (1972) results on the accumulation of cytokinin in the laminae of rooted bean leaves has confirmed her observations that the cytokinin produced by this system was a hitherto unidentified compound. To obtain sufficient of this compound for structure determination, we have used deca-
a
ZR
5.0-
2.584
10' 300
20 600
b
Z9G i
30 F No 900 E Vol
'~ 10 KINETIN
ZR i
i
~5.O
u.. Q. 2.5
(.2_
Treatment with [3-Glucosidase Samples for treatment were dissolved in Solvent E and dried into small vials. They were then incubated with 25 lag of/~-glucosidase (Boehringer Corp. Ltd.) in 0.1 mol dm -3 sodium acetate buffer, pH 5.3, overnight at 37 ~ C. After drying, samples were removed from the vial using 10 lal of ethanol followed by 10 lal 80% ethanol (v/v).
Glucose Detection Samples for glucose assay were treated with /~-glucosidase as described. The residue was dissolved in 25 gl Solvent E and chromatographed in Solvent B on paper. The glucose was visualised using " G l u c o s t a t " reagent (Sigma Chemical Co. Ltd.).
~o'
soo
2o' 6oo
so F.o '~ ~o 90o Ev,~ KI.ET~.
Fig. 1. a Tobacco callus bioassay of bean leaf extract chromatographed on Sephadex LH 20. A 2.5 x 75 column was eluted with Solvent A at a flow rate of 30 cm 3 h - 1 30 cm 3 fractions were collected and 0.1 cm 3 (equivalent to about 4 g fresh weight of tissue) withdrawn for assay. Fractions 11 and 12 were combined and used for further purification, b Tobacco callus assay of bean leaf extract chromatographed on Sephadex LH 20. A 2.5 x 72 cm column was eluted with Solvent B at a flow rate of 30 c m ? h 1.30 cm 3 fractions were collected and 0.1 cm 3 withdrawn for assay. Fractions 20 and 21 were combined and used for paper chromatography
T.L. Wang et al.: A Cytokinin Glucoside from Leaves
287
KINETIN~gl I
and similarly chromatographed. The results indicated that the unknown cytokinin was degraded by/%glucosidase to a biologically active compound or compounds which chromatographed with zeatin in Solvent F. Thus it was concluded that the unknown cytokinin was probably a /LD-glucoside. Permanganate treatment of further aliquots, after enzymic removal of the glucose moiety and chromatography in Solvent D followed by bioassay, showed that the aglycone was resistant to such oxidation. Hence the compound did not possess an isopentenyl side chain. Final purification of the compound was achieved by paper chromatography in Solvents C and F. An ultraviolet spectrum of the purified compound in Solvent E gave a •max at 269 nm while, at high pH (ammoniacal Solvent E), a shift to 275 nm with a shoulder at 284 nm was observed. Such a spectrum is characteristic of N6-substituted purines with no other ring substituents. From the absorption obtained approximately 40 ~tg kg- 1 of the purified compound was isolated (based on an em,xof 14.6 x 1 0 3 for zeatin). The purified compound was hydrolysed with /% glucosidase and the glucose released positively identified using glucose oxidase (Glucostat). The aglycone was subjected to GCMS and preparative GLC as the TMS derivative. GCMS of the aglycone gave the following ions at m/e ratios of: M § 365 (12.1), 350 (19.3), 276 (3.4), 275 (4.1), 274 (2.3), 262 (9.6), 260 (5.5), 236 (6.5), 235 (32.4), 234 (100), 233 (4.8), 232 (5.0), 222 (6.5), 221 (25.5), 220 (73.4), 193 (4.7), 192 (12.1), 162 (5.7) and it was therefore identified as dihydrozeatin. Preparative GLC followed by bioassay showed that all the cytokinin activity in the sample was confined to the dihydrozeatin-TMS peak (Fig. 2). On the basis of the above results, the major cytokinin in the leaves of decapitated bean plants was tentatively identified as 6-(4-O-fl-D-glucosyl-3-methylbutylamino) purine (dihydrozeatin-O-/~-D-glucoside). The identification of this compound, although not confirmed by synthesis, represents the most positive proof to date of the occurrence of endogenous cytokinin glucosides in higher plants.
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0
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