Planta (Berl.) 124, 207--210 (1975) 9 by Springer-Verlag 1975
A Quantitative Analysis of Cytokinin Using Single-Ion-Current-Monitoring A. G. T h o m p s o n , R. H o r g a n , a n d J. K . H e a l d Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, U. K. Received 15 February; accepted 21 March 1975
Summary. The levels of the eytokinin 6-(o-hydroxybenzylamino)-9-fl-D-ribofuranosylpurine (o-OH BAP riboside) have been measured in attached leaves of poplar (Populus • robusta Sehneid) using the technique of single-ion current monitoring (SICM). The use of 6-(p-hydroxybenzylamino)-9-f~-D-ribofuranosylpurine (p-OH BAP riboside) as an internal standard has enabled quantitative measurements of recovery to be made.
Introduction
I t h a s been shown ( H e w e t t a n d W a r e i n g , 1973) t h a t a s h o r t light t r e a t m e n t c a n increase t h e e y t o k i n i n levels i n d e t a c h e d p o p l a r leaves. T h e c y t o k i n i n i n v o l v e d was l a t e r identified as o - O H B A P riboside ( t t o r g a n et al., 1973). Other evidence of s h o r t - t e r m light effects on c y t o k i n i n levels (Van S t a d e n a n d W a r e i n g , 1972) is available, b u t this d i d n o t p r o v i d e o p p o r t u n i t y for q u a n t i t a t i v e s t u d y . SICM has been used to o b t a i n q u a n t i t a t i v e e s t i m a t e s of G A ' s ( F r y d m a n et al., 1974) a n d A B A ( R a i l t o n et al., 1974) in higher p l a n t s a l t h o u g h i n t e r n a l s t a n d a r d s were n o t u s e d i n t h e s e studies. Materials and Methods Plant Material. Poplar 'stools" (Populus • robusta Sctmeid) cut back annually to provide a supply of vigorous one-year old shoots were used, and leaf samples taken from the easterly side of the "stools" in the region of the third to sixth fully expanded leaves from the apex. Conditions. Sunrise on 3rd July 1974 was at 04.40 British Standard Time and this was taken as time 0. There was light cloud cover for the day giving 8 h of sunshine. The maximum temperature was 15.7~ and the minimum was 12.0~ There was 3.4 ml of precipitation during the day. Extraction. After harvesting the leaves, 50 g per treatment, were frozen in liquid air, and extracted into 500 ml cold 70% methanol, containing 10.2 ~g p-OH BAP riboside. The extract was stirred at 4~ for 12 h and filtered. The residue was resuspended and refiltered twice and the combined filtrates reduced to aqueous under vacuum at 30~ The volume was adjusted to 200 ml and the pH to 2.5 with dilute hydrochloric acid. The extract was frozen at --20 ~C, thawed and centrifuged at 23000 • g for 90 rain to remove lipid material. The supernatant was passed through a 2 • cm column of Zerolit 225 (H+ form) which was washed with 100 ml water at p i t 3.5 followed by 100 ml 70% methanol. The column was eluted with 500 ml of 2N NH4OH. The ammoniacal eluate was reduced to 150 ml on a rotary evaporator at 30~ the pH adjusted to 8.1 and partitioned 4• with equal volumes of water-saturated n-butanol. The butanol extracts were reduced to dryness on a rotary evaporator at 30~ and the residue
Abbreviations: ABA--Abscisic Acid, B.S.A.---Bis-(trimethylsilyl)-aeetamide,GA's--Gibberellins, o-OH BAP riboside--6-(o-hydroxybenzylamino)-9-/~-D-ribofuranosylpurine, SICM-Single Ion Current Monitoring, T.M.S.--Tri-methyl-silyl. 14"
208
A.. G. Thompson et al.
dissolved in 150 ml of water at pH 8.1. This solution was then extracted 5 X with equal volumes of ethyl acetate. The ethyl acetate was evaporated to dryness, the residue dissolved in 5 ml of Solvent A and loaded onto a 2 • 20 cm cellulose powder column packed in the same solvent. The column was eluted with 300 ml of Solvent A and the eluate was reduced to dryness and the residue taken up in 1.5 ml 35% ethanol. Insoluble material was removed by eentrifugation at 1000 x g for 20 min and the supernatant applied to a LH-20 Sephadex column using 35% ethanol as the elution solvent. The elution volume of o-OH BAP riboside had already been determined using synthetic material and this volume was collected from the plant sample. The 35% ethanol eluate was reduced to dryness and the residue transferred to a melting point capillary in 50 Izl of hot ethanol. The ethanol was removed under vacuum and the sample dried in vacuo over t)205 for 12 h. The T ~ S derivatives were prepared by heating the sample with 25 ~zl of B.S.A. at 80~ for 60 min. GLC was carried out using 5 ft • 4 mm I.C. (1520 • 4 mm) 2 % OV 1 columns at an isothermal temperature of 258 ~C with a carrier flow rate of 40 ml/min. Single ion current monitoring (SICM) was carried out using the parent ion of TMS 6-(0 or p-hydroxybenxylamino)-9-fl-D-ribofuranosylpurine at M/E 661 (Horgan, Hewett, Purse and Wareing, 1973). High frequency noise was eliminated by a suitable filter circuit adjusted to give minimum peak distortion. Solvent A; Butan-2-ol: 25% NH4OH (4:1 by volume)
Results and Discussion The aim of the work presented here was to verify the results of Hewett and Wareing 1973 and to obtain reliable quantitative results of the changes in levels of o-OH BAP riboside. A short time course of up to 6 h was chosen for convenience. The results indicated in Fig. 1 show the amount of o-OH BAP riboside in tzg per 50 g fresh weight of leaf tissue. Fig. 2 shows the SICM trace showing para and ortho peaks compared with total ion current trace for the same sample. The results show the rapid increase in o-OH BAP riboside as indicated by Hewett and Wareing 1973, but found the maximum value at only 5 rain after daybreak compared with 35 rain in detached leaves after red light treatment.
1.0
o
0.9
>
~07 o ~06
"c.._ a. ~ 0.5 .c 04 o
~
o~
>-03
o2 0"1 0
3'0
60
90
120
150
180
210 240 270 300 330 3'60
Time from dawn [mini
Fig. 1. The levels of o-OH BAt) riboside present in 50 g of fresh weight leaves at periods throughout the day. A calculated analytical error of 10% is present in each of the readings
Quantitative Analysis of Cytokinin
P
209
Single-ion current
Total-ion current
-6-
Time
Fig. 2. Comparison of the single-ion peaks m/e 661 for one of the samples to the total-ion peaks for the same sample shown on a GLC run. P p-OH BAP riboside; 0 o-OH BAP riboside
I~ote must be taken t h a t this does not consider effective light radiation apparent before 04.40 BST the initial time taken in this experiment. No evidence is available to determine whether the sudden increase is a release or a synthesis of o-OH BAP riboside this problem is at present being investigated. The pattern of change in the level of o-OH BAP riboside over the 6 h period investigated suggests the possibility of some form of internal rhythm. Hewett and Wareing 1973 have indicated the presence of a diurnal r h y t h m for the total eytokinin content of attached poplar leaves using paper chromatography and bioassay. Experiments are in progress to further investigate the rhythmic feature feature using SICM. The physiological significance of the increase in cytokinin is not yet understood as no clear evidence of the physiological role of o-OH BAP riboside in poplar has yet been found. However the rather spectacular control by light of the levels of this unusual compound in poplar leaves merits further investigation. We wish to thank the S.R.C. for a studentship to one of us, A.G.T., and the grant to purchase the GCMS.
210
A.G. Thompson et al. References
Frydman, V.M., Gaskin, P., MacMillan, J.: Qualitative and quantitative analysis of gibberellins throughout seed maturation in Pisum sativum ev Progress No. 9. Planta (Berl.) 118, 123-132 (1974) Hewett, E. W., Wareing, P. 1~.: Cytokinins in Populus X robusta (Schneid): Light effects on endogenous levels. Planta (Berl.) 114, 119-129 (1973) Horgan, R., Hewett, E.W., Purse, J. G., Wareing, P . F . : A new cytokinin from Populus robusta. Tetrahedron Letters 80, 2827-2828 (1973) Railton, I. D., Reid, D. M., Gaskin, P., MacMillan, J. : Charaeterisation of abseisie acid in chloroplasts of Pisum sativum L cv Alaska by combined gas-chromatography massspectroscopy. Planta (Berl.) 117, 179-182 (1974) Staden, J. van, Wareing, P. F. : The effect of light on endogenous eytokinin levels in seeds of Rumex obtusi/olius. Planta (Berl.) 194, 126-133 (1972)
A Quantitative Analysis of Cytokinin Using Single-Ion-Current-Monitoring A. G. T h o m p s o n , R. H o r g a n , a n d J. K . H e a l d Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, U. K. Received 15 February; accepted 21 March 1975
Summary. The levels of the eytokinin 6-(o-hydroxybenzylamino)-9-fl-D-ribofuranosylpurine (o-OH BAP riboside) have been measured in attached leaves of poplar (Populus • robusta Sehneid) using the technique of single-ion current monitoring (SICM). The use of 6-(p-hydroxybenzylamino)-9-f~-D-ribofuranosylpurine (p-OH BAP riboside) as an internal standard has enabled quantitative measurements of recovery to be made.
Introduction
I t h a s been shown ( H e w e t t a n d W a r e i n g , 1973) t h a t a s h o r t light t r e a t m e n t c a n increase t h e e y t o k i n i n levels i n d e t a c h e d p o p l a r leaves. T h e c y t o k i n i n i n v o l v e d was l a t e r identified as o - O H B A P riboside ( t t o r g a n et al., 1973). Other evidence of s h o r t - t e r m light effects on c y t o k i n i n levels (Van S t a d e n a n d W a r e i n g , 1972) is available, b u t this d i d n o t p r o v i d e o p p o r t u n i t y for q u a n t i t a t i v e s t u d y . SICM has been used to o b t a i n q u a n t i t a t i v e e s t i m a t e s of G A ' s ( F r y d m a n et al., 1974) a n d A B A ( R a i l t o n et al., 1974) in higher p l a n t s a l t h o u g h i n t e r n a l s t a n d a r d s were n o t u s e d i n t h e s e studies. Materials and Methods Plant Material. Poplar 'stools" (Populus • robusta Sctmeid) cut back annually to provide a supply of vigorous one-year old shoots were used, and leaf samples taken from the easterly side of the "stools" in the region of the third to sixth fully expanded leaves from the apex. Conditions. Sunrise on 3rd July 1974 was at 04.40 British Standard Time and this was taken as time 0. There was light cloud cover for the day giving 8 h of sunshine. The maximum temperature was 15.7~ and the minimum was 12.0~ There was 3.4 ml of precipitation during the day. Extraction. After harvesting the leaves, 50 g per treatment, were frozen in liquid air, and extracted into 500 ml cold 70% methanol, containing 10.2 ~g p-OH BAP riboside. The extract was stirred at 4~ for 12 h and filtered. The residue was resuspended and refiltered twice and the combined filtrates reduced to aqueous under vacuum at 30~ The volume was adjusted to 200 ml and the pH to 2.5 with dilute hydrochloric acid. The extract was frozen at --20 ~C, thawed and centrifuged at 23000 • g for 90 rain to remove lipid material. The supernatant was passed through a 2 • cm column of Zerolit 225 (H+ form) which was washed with 100 ml water at p i t 3.5 followed by 100 ml 70% methanol. The column was eluted with 500 ml of 2N NH4OH. The ammoniacal eluate was reduced to 150 ml on a rotary evaporator at 30~ the pH adjusted to 8.1 and partitioned 4• with equal volumes of water-saturated n-butanol. The butanol extracts were reduced to dryness on a rotary evaporator at 30~ and the residue
Abbreviations: ABA--Abscisic Acid, B.S.A.---Bis-(trimethylsilyl)-aeetamide,GA's--Gibberellins, o-OH BAP riboside--6-(o-hydroxybenzylamino)-9-/~-D-ribofuranosylpurine, SICM-Single Ion Current Monitoring, T.M.S.--Tri-methyl-silyl. 14"
208
A.. G. Thompson et al.
dissolved in 150 ml of water at pH 8.1. This solution was then extracted 5 X with equal volumes of ethyl acetate. The ethyl acetate was evaporated to dryness, the residue dissolved in 5 ml of Solvent A and loaded onto a 2 • 20 cm cellulose powder column packed in the same solvent. The column was eluted with 300 ml of Solvent A and the eluate was reduced to dryness and the residue taken up in 1.5 ml 35% ethanol. Insoluble material was removed by eentrifugation at 1000 x g for 20 min and the supernatant applied to a LH-20 Sephadex column using 35% ethanol as the elution solvent. The elution volume of o-OH BAP riboside had already been determined using synthetic material and this volume was collected from the plant sample. The 35% ethanol eluate was reduced to dryness and the residue transferred to a melting point capillary in 50 Izl of hot ethanol. The ethanol was removed under vacuum and the sample dried in vacuo over t)205 for 12 h. The T ~ S derivatives were prepared by heating the sample with 25 ~zl of B.S.A. at 80~ for 60 min. GLC was carried out using 5 ft • 4 mm I.C. (1520 • 4 mm) 2 % OV 1 columns at an isothermal temperature of 258 ~C with a carrier flow rate of 40 ml/min. Single ion current monitoring (SICM) was carried out using the parent ion of TMS 6-(0 or p-hydroxybenxylamino)-9-fl-D-ribofuranosylpurine at M/E 661 (Horgan, Hewett, Purse and Wareing, 1973). High frequency noise was eliminated by a suitable filter circuit adjusted to give minimum peak distortion. Solvent A; Butan-2-ol: 25% NH4OH (4:1 by volume)
Results and Discussion The aim of the work presented here was to verify the results of Hewett and Wareing 1973 and to obtain reliable quantitative results of the changes in levels of o-OH BAP riboside. A short time course of up to 6 h was chosen for convenience. The results indicated in Fig. 1 show the amount of o-OH BAP riboside in tzg per 50 g fresh weight of leaf tissue. Fig. 2 shows the SICM trace showing para and ortho peaks compared with total ion current trace for the same sample. The results show the rapid increase in o-OH BAP riboside as indicated by Hewett and Wareing 1973, but found the maximum value at only 5 rain after daybreak compared with 35 rain in detached leaves after red light treatment.
1.0
o
0.9
>
~07 o ~06
"c.._ a. ~ 0.5 .c 04 o
~
o~
>-03
o2 0"1 0
3'0
60
90
120
150
180
210 240 270 300 330 3'60
Time from dawn [mini
Fig. 1. The levels of o-OH BAt) riboside present in 50 g of fresh weight leaves at periods throughout the day. A calculated analytical error of 10% is present in each of the readings
Quantitative Analysis of Cytokinin
P
209
Single-ion current
Total-ion current
-6-
Time
Fig. 2. Comparison of the single-ion peaks m/e 661 for one of the samples to the total-ion peaks for the same sample shown on a GLC run. P p-OH BAP riboside; 0 o-OH BAP riboside
I~ote must be taken t h a t this does not consider effective light radiation apparent before 04.40 BST the initial time taken in this experiment. No evidence is available to determine whether the sudden increase is a release or a synthesis of o-OH BAP riboside this problem is at present being investigated. The pattern of change in the level of o-OH BAP riboside over the 6 h period investigated suggests the possibility of some form of internal rhythm. Hewett and Wareing 1973 have indicated the presence of a diurnal r h y t h m for the total eytokinin content of attached poplar leaves using paper chromatography and bioassay. Experiments are in progress to further investigate the rhythmic feature feature using SICM. The physiological significance of the increase in cytokinin is not yet understood as no clear evidence of the physiological role of o-OH BAP riboside in poplar has yet been found. However the rather spectacular control by light of the levels of this unusual compound in poplar leaves merits further investigation. We wish to thank the S.R.C. for a studentship to one of us, A.G.T., and the grant to purchase the GCMS.
210
A.G. Thompson et al. References
Frydman, V.M., Gaskin, P., MacMillan, J.: Qualitative and quantitative analysis of gibberellins throughout seed maturation in Pisum sativum ev Progress No. 9. Planta (Berl.) 118, 123-132 (1974) Hewett, E. W., Wareing, P. 1~.: Cytokinins in Populus X robusta (Schneid): Light effects on endogenous levels. Planta (Berl.) 114, 119-129 (1973) Horgan, R., Hewett, E.W., Purse, J. G., Wareing, P . F . : A new cytokinin from Populus robusta. Tetrahedron Letters 80, 2827-2828 (1973) Railton, I. D., Reid, D. M., Gaskin, P., MacMillan, J. : Charaeterisation of abseisie acid in chloroplasts of Pisum sativum L cv Alaska by combined gas-chromatography massspectroscopy. Planta (Berl.) 117, 179-182 (1974) Staden, J. van, Wareing, P. F. : The effect of light on endogenous eytokinin levels in seeds of Rumex obtusi/olius. Planta (Berl.) 194, 126-133 (1972)
