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A rapid and sensitive assay for abscisic acid using ethyl abscisate as an internal standard
ANALYTICAL
BIOCHEMISTRY
87, 148- 156 (1978)
A Rapid and Sensitive Ethyl Abscisate
Assay for Abscisic Acid Using as an internal Standard S. A. QUARRIE
Plant
Breeding
Institute.
Maris
Lane.
Trumpington,
Cambridge
CB2 2LQ.
United
Kingdom
Received June 6, 1977: accepted January 26, 1978 A gas-liquid chromatographic (glc) assay for abscisic acid (ABA) is described that is both rapid and highly sensitive. The selectivity and sensitivity of an electroncapture detector are used to reduce the assay procedure to just four steps: extraction, thin-layer chromatography (tic), esterification, and glc. Quantification of the ABA present is by reference to ethyl abscisate added during the assay as an internal standard. The procedure enables samples containing as little as 100 pg of ABA to be assayed.
The presence of abscisic acid (ABA) in plant tissues is known to vary considerably with plant water status (l-3). In an investigation to compare the production of abscisic acid in response to water stress within a wide range of wheat genotypes, it was required to carry out a large number of ABA assays. Using conventional purification procedures (4,5) this would have taken an excessive time. Therefore an assay for ABA was needed that was quick yet reliable. There have been recent attempts to speed up the assay procedures for ABA either by modification of the solvent partitioning stages (3) or by use of high-performance liquid chromatography for the combined purification and quantification of the free acid (6). However, these modifications still involve a number of time-consuming steps and losses of ABA are likely to occur. The report by Seeley and Powell (7) that low levels of ABA as its methyl ester may be readily detected by an electron-capture detector (ECD) with gas chromatography has led to the use of ECDs by several workers for the quantification of ABA (2,3,8,9). However, the sensitivity and selectivity of this type of detector for ABA have not yet been fully exploited for the measurement of the hormone. This paper describes an assay for ABA that relies on these two properties of the ECD to enable many of the lengthy purification stages to be omitted and hence to reduce the losses of ABA during the assay and to reduce the sample size necessary for each assay.
0003-2697/78/0871-0148$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
RAPID
ASSAY
MATERIALS
FOR
ABSCISIC
149
ACID
AND METHODS
Apparatus. Extraction of the leaf tissue was by ultrasonic disintegration using a Dawe Instruments, Ltd., Soniprobe Type 7532A fitted with a microtip. Gas chromatography was carried out with a Pye Unicam, Ltd., gas chromatograph Model 104 fitted with an ECD containing a 63Ni foil. Operation of the detector was either in the pulse-spaced mode (150 psec, attenuator 100, 1 x IO-lo A full-scale deviation) or, more usually, in the pulse-modulated mode by connection to a Pye GCV electron-capture amplifier module (range 8 or 16). A glass column, 150 x 0.4 cm, containing 1.2% SE30 + 0.3% XE60 on 80- to loo-mesh Diatomite CLQ was used isothermally at 197°C with the detector oven temperature at 225°C. Nitrogen was used as the carrier gas, the flow rate being 75 cm3 min-‘. Peak areas were integrated using an Infotronics Model CRS 309 integrator. Reagents. Racemic cis -trans-ABA’ was obtained from Sigma Chemical Co., and racemic [2-14C]ABA (6.1 mCi*mmol-I), from the Radiochemical Centre (Amersham, U.K.). All solvents were either analytical or Distol (cyclohexane) grade and were distilled before use only where stated. The glassware used in each assay was cleaned with Decon 90 decontamination fluid (Decon Laboratories, Ltd.). Stationary phases and support materials for gas chromatography were obtained from J.J.‘s (Chromatography), Ltd. Procedure. The overall scheme for extraction of plant material and assay of ABA is illustrated in Fig. 1. Fresh plant material, usually 0.1-0.3 g of lamina from a single wheat leaf, was cut into 2- to 3-mm sections and stored at -30°C until required. Extracts were prepared in the dark at 0°C by ultrasonic disintegration of the leaf segments in acetone:water (9: 1, v/v) using a solvent volume to leaf fresh weight (FW) ratio of 10: 1 (cubic centimeters:grams). Extraction was deemed complete upon removal of the chlorophyll from the tissues. This normally took 5 min at setting 3 (50 W), though the sonication time was longer when the leaf material was severely drought stressed. The extracts could be used immediately but were normally allowed to settle in the refrigerator until required, generally within 48 hr of sonication. Samples of the supernatant from the crude aqueous acetone extract were purified by thin-layer chromatography (tic) using Merck silica gel GF,,, precoated analytical tic plates cut to 5 x 5 cm. An aliquot of 300 ,ul of extract (equivalent to 30 mg FW of leaf) was applied to the base of each plate, which was subsequently developed in ethyl acetate in a chromatography tank saturated with solvent vapour. Volumes larger than 300 ~1 tended to overload the plates and affect the running characteristics. ’ Cis-truns-ABA MeABA: and ethyl
will subsequently cis-frans-abscisate,
be referred to as ABA: as EtABA.
methyl
cis-rrans-abscisate,
as
150
S. A. QUARRIE Plant material I Sonication in 90% acetone
I
tic in ethyl acetate EtABA added as internal standard I Esterification with diazomethane
I
glc with electron-capture
detector
FIG. 1. Outline of assay procedure.
The position of the ABA band was determined by comparison with a reference plate obtained by chromatographing an extract containing sufficient added ABA for visible quenching of uv fluorescence. In this solvent the R, of the ABA band was stable relative to coloured carotenoid bands, one of which ran slightly ahead of the ABA band. This carotenoid band was subsequently used as an internal reference. tvans-trans-ABA ran as a distinct band ahead of the ABA and therefore did not interfere with the assay. A 4-mm-wide band of silica containing the ABA was removed from each plate and the ABA was eluted into a 300~~1 conical vial with 2 x 175 ~1 of water-saturated distilled ethyl acetate (10) using controlled suction. Ethyl abscisate was prepared from ABA and diazoethane, which was obtained in 40-60°C petroleum ether solution* from nitrosoethylurea [made from ethylamine by the procedure given in Ref. (1 l)] and a 40% potassium hydroxide solution. A 2 x lo-’ M solution of EtABA in cyclohexane was added to the eluate to act as an internal standard for the subsequent steps of the analysis. Sufficient EtABA solution was added to give a MeABA: EtABA ratio usually between 0.5: 1 and 2: 1. For most assays of waterstressed leaves the amount added was 50 (2.92 ng) or 100 ~1 (5.84 ng). Esterification of the ABA was achieved with diazomethane, which was prepared in distilled 40-60°C petroleum ether using nitrosomethylurea prepared from methylamine hydrochloride (11) and 40% potassium hydroxide solution. To remove traces of entrained potassium hydroxide it was necessary to distil the diazomethane twice into fresh distilled 40-60°C petroleum ether. Fifty microliters of a weak diazomethane solution (pale yellow) was added to the ABA solution, which was left in the dark until the reaction was complete. After 5 min the solvents were removed by evaporation under a stream of oxygen-free nitrogen. The residue was taken up in cyclohexane, typically 100 ~1, and 2 ~1 of this solution was injected into 2 Explosion hazard: Diazoethane is less stable than diazomethane and the petroleum ether solution of diazoethane was therefore used without further purification.
RAPID ASSAY FOR ABSCISIC
/
0
151
ACID
EIABA
4
8 min.
0
4
8 min.
4
8 mln
FIGS. 2a and b (left and center). Duplicate assays of a water-stressed wheat leaf extract; 5.84 ng of EtABA was added to each assay. EtABA peak represents 11.50of total EtABA present (117 pg). FIG. 3 (right). Assay of silica gel from a clean tic plate after development in ethyl acetate. No EtABA added, but treated with diazomethane.
the gas-liquid chromatograph (glc). The ratios of the areas for MeABA and EtABA were used to calculate the ABA content (nanograms per gram FW) of the leaf. A sample of each leaf was oven dried to obtain the dry weight: fresh weight ratio. For routine assays a single extraction of each leaf was carried out and the resulting extract was assayed twice. The duplicate assays usually gave values within 25% of the mean when a clean ECD was used. RESULTS AND DISCUSSION
Typical assay results obtained with the gas chromatograph are illustrated in Fig. 2. The major contaminants following the initial peak came from the tic plates (Fig. 3), though the size of these peaks could have been con-
152
S. A. QUARRIE
&ABA
\
0
4
8 min.
0’
4
8 min.
FIG. 4 (left). Assay of unstressed wheat leaf extract without EtABA added. Assay volume and glc amplification range the same as in Figs. 2a and b. FIG. 5 (right). Assay solution of stressed wheat leaf extract used in Fig. 2a after treating 20 ~1 with long-wave (366 nm) uv light for 40 min.
siderably enhanced by contamination arising elsewhere in the assay. The choice of chromatograph column stationary phase proved critical to give short analysis times, to obtain good resolution of EtABA and MeABA, and to separate these two components from the tic plate impurities. There were no other peaks present from any of the plant extracts used which interfered with the ABA ester peak (Fig. 4). Because of the limited purification involved in the assay several checks were made to verify the authenticity and purity of the MeABA peak. The identity of the assay peak with MeABA was presumed from the following results. (i) The assay esters always cochromatographed with authentic MeABA using a wide polarity range of stationary phases (SE30, SE54. OVl, OVlOl, OV17,OV210, XE60, and binary combinations of these) and on no occasion was the MeABA peak resolved beyond a single component. (ii) Treatment of the assay samples with diazoethane instead of diazo-
RAPID ASSAY FOR ABSCISIC TABLE EFFECT
OF RECHROMATOGRAPHY SOLVENT
Ethyl acetate:cyclohexane (60:40) Chloroform:methanol (96:4) Diethyl ether:carbon tetrachloride (90: 10) Toluene:methanoI (80:20) n MeABA:EtABA
I
OF ASSAY
ESTERS
SYSTEMS ON THE MeABA:EtABA
Solvent system (v/v)
153
ACID
IN DIFFERENT RATIO”
MeABA:EtABA
ratio
Initial
Final
0.45-0.50
0.80
0.78
0.41-0.49
0.80
0.85
0.43-0.49
0.99
1.04
0.43-0.45
0.68
0.67
Ester R, (range)
ratios were obtained with 5.84 ng of EtABA present.
methane gave a single peak with the same retention time as authentic EtABA and, with MeABA incorporated as the internal standard, the EtABA:MeABA ratio was identical to that obtained using the normal procedure. (iii) b-radiation of the assay esters in cyclohexane with long-wave uv light to give a mixture of cis-tram and truns -tram esters (4) (Fig. 5) never altered the MeABA:EtABA ratio. (iv) The ratio of MeABA:EtABA was never significantly altered by thinlayer rechromatography of the esters in several solvent systems (Table 1). (v) The size of the MeABA peaks was consistent with the value expected from the water potential of the leaf from which the sample was taken, i.e., ABA content increased as the leaf water potential fell. As there were few steps involved from the plant to the gas chromatograph the losses of ABA were small. To investigate the reproducibility of the extraction by sonication, two stressed wheat leaves were cut up, mixed, and subsampled to give five samples offresh weight 0.1-0.3 g. Each was sonicated and assayed twice (Table 2). The five extracts gave a mean MeABA:EtABA ratio of 0.670 2 0.017 (-tSE). These data show that extraction efficiency was not affected by sample size. Reextraction of sonicated material gave on the average a further 10% of ABA (Table 3). Using plant extracts containing added cis -~rans-[2-14C]ABA recovery of labelled ABA from thin-layer plates after development was calculated to be 89.6 * 1.9%. Further radioactivity from the ABA zone could be recovered by repeated elution of the silica. To check the reproducibility of the assay synthetic ABA was added to an extract of unstressed wheat leaves, which, for quantitative recovery of the ABA added, would give an MeABA:EtABA ratio of 1.20. This extract was assayed in duplicate five times, and the five mean MeABA:EtABA ratios varied from 1.05 to 1. Il. The overall mean of I .08 represented a recovery of 90.0% from the thin-layer plates. These
154
S. A. QUARRIE TABLE EFFECT
OF SAMPLE
2
SIZE ON THE ABA
MeABA:EtABA
EXTRACTED”
ratio
Sample FW (b9
(a)
(b)
Mean
Mean ABA content (ngig FW
0.10 0.15 0.20 0.25 0.30
0.62 0.62 0.72 0.66 0.61
0.70 0.64 0.73 0.71 0.69
0.66 0.63 0.73 0.69 0.65
71 68 79 74 70
a MeABA:EtABA
ratios were obtained with 2.92 ng of EtABA present.
data for sonication and elution efficiencies represent a mean recovery of MeABA from readily extractable ABA in the plant tissues of 81%. The reliability of the assay was found to depend largely upon the state of the ECD, which, because of the limited purification of each sample, would tend to become contaminated fairly readily. However, with carefully controlled operating conditions it was found that a detector could be used for about 1000 injections before its response became unreliable, necessitating cleaning of the radioactive source by the manufacturers. The process time for each leaf extract during continuous assaying was 40 min (20 min/assay), and the sensitivity of a clean detector was such that a tissue sample containing as little as 100 pg of ABA could be assayed reliably. This value represents a tissue fresh weight of less than a milligram for wheat leaves at a potential of - 12 to - 14 bar. For low tissue concentrations of ABA the sensitivity and accuracy of the assay were governed by the amount of material that could be applied to the thin-layer plate. In such cases it may be necessary to use a prepurification step to improve sensitivity. Although the assay was developed primarily to speed up routine determinations of ABA in water-stressed wheat leaves, the TABLE AMOUNTS
Initial extract MeABA:EtABA ratio
Resonicated extract MeABA:EtABA ratio
1
2
Mean
1.06 0.80 0.86
0.97 0.74 0.87
1.01
0.10
0.77 0.87
” MeABA:EtABA
3
OF ABA EXTRACTED INITIALLY FROM STRESSED LEAVES ON REEXTRACTION OF THE CENTRIFUGED RESIDUES”
AND
ABA (% of that initially extracted)
2
Mean
0.07
0.09 0.07
0.09 0.07
9 9
0.10
0.10
0.10
12
1
ratios were obtained with 2.92 ng of EtABA present.
155
RAPID ASSAY FOR ABSCISIC ACID
EtABA
,,,,,( *b, /
J
0
4 min.
a
0
4 rnln.
8
FIG. 6 (left). Assay of 3.5 mg of xylem sap collected from the cut surface ofa stressed wheat leaf under pressure in a pressure bomb; 1.17 ng of EtABA was added to the assay. EtABA peak represents l/20 of total EtABA present (58 pg). MeABA:EtABA ratio represents 205 ng of ABA/ml of xylem sap. FIG. 7 (right). Assay of 16 mg of lower epidermis removed from a stressed leaf of Vicinfuba; 1.17 ng of EtABA was added to the assay. MeABA:EtABA ratio represents 110 ng of ABA/g FW of epidermis.
sensitivity of the technique renders it ideal for studies of some of the fundamental questions concerning the physiological role of ABA in controlling stomata1 movements and other responses. For example, the ABA concentration in expressed xylem sap (Fig. 6) and in isolated leaf epidermis (Fig. 7) has been measured. ACKNOWLEDGMENT The expert technical assistance of Miss Pam Smith in the development of the assay and in its routine use is gratefully acknowledged.
156
S. A. QUARRJE
REFERENCES I. 2. 3. 4. 5. 6. 7. 8.
Wright, S. T. C. (1969) Planta 86, 10-20. Zabadal, T. J. (1974) Plant Physiol. 53, 125-127. Beardsell, M. F., and Cohen, D. (1975) Plant Physiol. 56, 207-212. Lenton, J. R., Perry, V. M., and Saunders, P. F. (1971) Planta 96, 271-280. Colquhoun, A. J., and Hillman, J. R. (1975) Z. PJanzenphysiol. 76, 326-332. Sweetser, P. B., and Vatvars, A. (1976) Anal. Biochem. 71, 68-78. Seeley, S. D., and Powell, L. E. (1970) Anal. Biochem. 35, 530-533. Mizrahi, Y., Blumenfeld, A., Bittner, S., and Richmond, A. E. (1971) Plant Physiol. 48, 752-755. 9. Loveys, B. R., and Kriedeman, P. E. (1974) Aust. .f. Planf Physiol. 1, 407-415. 10. Zeevaart, J. A. D. (1971) Plant Physiol. 48, 86-90. 11. Vogel, A. I. (1956) “Practical Organic Chemistry,” 3rd ed., p. 969. Longmans, London
BIOCHEMISTRY
87, 148- 156 (1978)
A Rapid and Sensitive Ethyl Abscisate
Assay for Abscisic Acid Using as an internal Standard S. A. QUARRIE
Plant
Breeding
Institute.
Maris
Lane.
Trumpington,
Cambridge
CB2 2LQ.
United
Kingdom
Received June 6, 1977: accepted January 26, 1978 A gas-liquid chromatographic (glc) assay for abscisic acid (ABA) is described that is both rapid and highly sensitive. The selectivity and sensitivity of an electroncapture detector are used to reduce the assay procedure to just four steps: extraction, thin-layer chromatography (tic), esterification, and glc. Quantification of the ABA present is by reference to ethyl abscisate added during the assay as an internal standard. The procedure enables samples containing as little as 100 pg of ABA to be assayed.
The presence of abscisic acid (ABA) in plant tissues is known to vary considerably with plant water status (l-3). In an investigation to compare the production of abscisic acid in response to water stress within a wide range of wheat genotypes, it was required to carry out a large number of ABA assays. Using conventional purification procedures (4,5) this would have taken an excessive time. Therefore an assay for ABA was needed that was quick yet reliable. There have been recent attempts to speed up the assay procedures for ABA either by modification of the solvent partitioning stages (3) or by use of high-performance liquid chromatography for the combined purification and quantification of the free acid (6). However, these modifications still involve a number of time-consuming steps and losses of ABA are likely to occur. The report by Seeley and Powell (7) that low levels of ABA as its methyl ester may be readily detected by an electron-capture detector (ECD) with gas chromatography has led to the use of ECDs by several workers for the quantification of ABA (2,3,8,9). However, the sensitivity and selectivity of this type of detector for ABA have not yet been fully exploited for the measurement of the hormone. This paper describes an assay for ABA that relies on these two properties of the ECD to enable many of the lengthy purification stages to be omitted and hence to reduce the losses of ABA during the assay and to reduce the sample size necessary for each assay.
0003-2697/78/0871-0148$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
RAPID
ASSAY
MATERIALS
FOR
ABSCISIC
149
ACID
AND METHODS
Apparatus. Extraction of the leaf tissue was by ultrasonic disintegration using a Dawe Instruments, Ltd., Soniprobe Type 7532A fitted with a microtip. Gas chromatography was carried out with a Pye Unicam, Ltd., gas chromatograph Model 104 fitted with an ECD containing a 63Ni foil. Operation of the detector was either in the pulse-spaced mode (150 psec, attenuator 100, 1 x IO-lo A full-scale deviation) or, more usually, in the pulse-modulated mode by connection to a Pye GCV electron-capture amplifier module (range 8 or 16). A glass column, 150 x 0.4 cm, containing 1.2% SE30 + 0.3% XE60 on 80- to loo-mesh Diatomite CLQ was used isothermally at 197°C with the detector oven temperature at 225°C. Nitrogen was used as the carrier gas, the flow rate being 75 cm3 min-‘. Peak areas were integrated using an Infotronics Model CRS 309 integrator. Reagents. Racemic cis -trans-ABA’ was obtained from Sigma Chemical Co., and racemic [2-14C]ABA (6.1 mCi*mmol-I), from the Radiochemical Centre (Amersham, U.K.). All solvents were either analytical or Distol (cyclohexane) grade and were distilled before use only where stated. The glassware used in each assay was cleaned with Decon 90 decontamination fluid (Decon Laboratories, Ltd.). Stationary phases and support materials for gas chromatography were obtained from J.J.‘s (Chromatography), Ltd. Procedure. The overall scheme for extraction of plant material and assay of ABA is illustrated in Fig. 1. Fresh plant material, usually 0.1-0.3 g of lamina from a single wheat leaf, was cut into 2- to 3-mm sections and stored at -30°C until required. Extracts were prepared in the dark at 0°C by ultrasonic disintegration of the leaf segments in acetone:water (9: 1, v/v) using a solvent volume to leaf fresh weight (FW) ratio of 10: 1 (cubic centimeters:grams). Extraction was deemed complete upon removal of the chlorophyll from the tissues. This normally took 5 min at setting 3 (50 W), though the sonication time was longer when the leaf material was severely drought stressed. The extracts could be used immediately but were normally allowed to settle in the refrigerator until required, generally within 48 hr of sonication. Samples of the supernatant from the crude aqueous acetone extract were purified by thin-layer chromatography (tic) using Merck silica gel GF,,, precoated analytical tic plates cut to 5 x 5 cm. An aliquot of 300 ,ul of extract (equivalent to 30 mg FW of leaf) was applied to the base of each plate, which was subsequently developed in ethyl acetate in a chromatography tank saturated with solvent vapour. Volumes larger than 300 ~1 tended to overload the plates and affect the running characteristics. ’ Cis-truns-ABA MeABA: and ethyl
will subsequently cis-frans-abscisate,
be referred to as ABA: as EtABA.
methyl
cis-rrans-abscisate,
as
150
S. A. QUARRIE Plant material I Sonication in 90% acetone
I
tic in ethyl acetate EtABA added as internal standard I Esterification with diazomethane
I
glc with electron-capture
detector
FIG. 1. Outline of assay procedure.
The position of the ABA band was determined by comparison with a reference plate obtained by chromatographing an extract containing sufficient added ABA for visible quenching of uv fluorescence. In this solvent the R, of the ABA band was stable relative to coloured carotenoid bands, one of which ran slightly ahead of the ABA band. This carotenoid band was subsequently used as an internal reference. tvans-trans-ABA ran as a distinct band ahead of the ABA and therefore did not interfere with the assay. A 4-mm-wide band of silica containing the ABA was removed from each plate and the ABA was eluted into a 300~~1 conical vial with 2 x 175 ~1 of water-saturated distilled ethyl acetate (10) using controlled suction. Ethyl abscisate was prepared from ABA and diazoethane, which was obtained in 40-60°C petroleum ether solution* from nitrosoethylurea [made from ethylamine by the procedure given in Ref. (1 l)] and a 40% potassium hydroxide solution. A 2 x lo-’ M solution of EtABA in cyclohexane was added to the eluate to act as an internal standard for the subsequent steps of the analysis. Sufficient EtABA solution was added to give a MeABA: EtABA ratio usually between 0.5: 1 and 2: 1. For most assays of waterstressed leaves the amount added was 50 (2.92 ng) or 100 ~1 (5.84 ng). Esterification of the ABA was achieved with diazomethane, which was prepared in distilled 40-60°C petroleum ether using nitrosomethylurea prepared from methylamine hydrochloride (11) and 40% potassium hydroxide solution. To remove traces of entrained potassium hydroxide it was necessary to distil the diazomethane twice into fresh distilled 40-60°C petroleum ether. Fifty microliters of a weak diazomethane solution (pale yellow) was added to the ABA solution, which was left in the dark until the reaction was complete. After 5 min the solvents were removed by evaporation under a stream of oxygen-free nitrogen. The residue was taken up in cyclohexane, typically 100 ~1, and 2 ~1 of this solution was injected into 2 Explosion hazard: Diazoethane is less stable than diazomethane and the petroleum ether solution of diazoethane was therefore used without further purification.
RAPID ASSAY FOR ABSCISIC
/
0
151
ACID
EIABA
4
8 min.
0
4
8 min.
4
8 mln
FIGS. 2a and b (left and center). Duplicate assays of a water-stressed wheat leaf extract; 5.84 ng of EtABA was added to each assay. EtABA peak represents 11.50of total EtABA present (117 pg). FIG. 3 (right). Assay of silica gel from a clean tic plate after development in ethyl acetate. No EtABA added, but treated with diazomethane.
the gas-liquid chromatograph (glc). The ratios of the areas for MeABA and EtABA were used to calculate the ABA content (nanograms per gram FW) of the leaf. A sample of each leaf was oven dried to obtain the dry weight: fresh weight ratio. For routine assays a single extraction of each leaf was carried out and the resulting extract was assayed twice. The duplicate assays usually gave values within 25% of the mean when a clean ECD was used. RESULTS AND DISCUSSION
Typical assay results obtained with the gas chromatograph are illustrated in Fig. 2. The major contaminants following the initial peak came from the tic plates (Fig. 3), though the size of these peaks could have been con-
152
S. A. QUARRIE
&ABA
\
0
4
8 min.
0’
4
8 min.
FIG. 4 (left). Assay of unstressed wheat leaf extract without EtABA added. Assay volume and glc amplification range the same as in Figs. 2a and b. FIG. 5 (right). Assay solution of stressed wheat leaf extract used in Fig. 2a after treating 20 ~1 with long-wave (366 nm) uv light for 40 min.
siderably enhanced by contamination arising elsewhere in the assay. The choice of chromatograph column stationary phase proved critical to give short analysis times, to obtain good resolution of EtABA and MeABA, and to separate these two components from the tic plate impurities. There were no other peaks present from any of the plant extracts used which interfered with the ABA ester peak (Fig. 4). Because of the limited purification involved in the assay several checks were made to verify the authenticity and purity of the MeABA peak. The identity of the assay peak with MeABA was presumed from the following results. (i) The assay esters always cochromatographed with authentic MeABA using a wide polarity range of stationary phases (SE30, SE54. OVl, OVlOl, OV17,OV210, XE60, and binary combinations of these) and on no occasion was the MeABA peak resolved beyond a single component. (ii) Treatment of the assay samples with diazoethane instead of diazo-
RAPID ASSAY FOR ABSCISIC TABLE EFFECT
OF RECHROMATOGRAPHY SOLVENT
Ethyl acetate:cyclohexane (60:40) Chloroform:methanol (96:4) Diethyl ether:carbon tetrachloride (90: 10) Toluene:methanoI (80:20) n MeABA:EtABA
I
OF ASSAY
ESTERS
SYSTEMS ON THE MeABA:EtABA
Solvent system (v/v)
153
ACID
IN DIFFERENT RATIO”
MeABA:EtABA
ratio
Initial
Final
0.45-0.50
0.80
0.78
0.41-0.49
0.80
0.85
0.43-0.49
0.99
1.04
0.43-0.45
0.68
0.67
Ester R, (range)
ratios were obtained with 5.84 ng of EtABA present.
methane gave a single peak with the same retention time as authentic EtABA and, with MeABA incorporated as the internal standard, the EtABA:MeABA ratio was identical to that obtained using the normal procedure. (iii) b-radiation of the assay esters in cyclohexane with long-wave uv light to give a mixture of cis-tram and truns -tram esters (4) (Fig. 5) never altered the MeABA:EtABA ratio. (iv) The ratio of MeABA:EtABA was never significantly altered by thinlayer rechromatography of the esters in several solvent systems (Table 1). (v) The size of the MeABA peaks was consistent with the value expected from the water potential of the leaf from which the sample was taken, i.e., ABA content increased as the leaf water potential fell. As there were few steps involved from the plant to the gas chromatograph the losses of ABA were small. To investigate the reproducibility of the extraction by sonication, two stressed wheat leaves were cut up, mixed, and subsampled to give five samples offresh weight 0.1-0.3 g. Each was sonicated and assayed twice (Table 2). The five extracts gave a mean MeABA:EtABA ratio of 0.670 2 0.017 (-tSE). These data show that extraction efficiency was not affected by sample size. Reextraction of sonicated material gave on the average a further 10% of ABA (Table 3). Using plant extracts containing added cis -~rans-[2-14C]ABA recovery of labelled ABA from thin-layer plates after development was calculated to be 89.6 * 1.9%. Further radioactivity from the ABA zone could be recovered by repeated elution of the silica. To check the reproducibility of the assay synthetic ABA was added to an extract of unstressed wheat leaves, which, for quantitative recovery of the ABA added, would give an MeABA:EtABA ratio of 1.20. This extract was assayed in duplicate five times, and the five mean MeABA:EtABA ratios varied from 1.05 to 1. Il. The overall mean of I .08 represented a recovery of 90.0% from the thin-layer plates. These
154
S. A. QUARRIE TABLE EFFECT
OF SAMPLE
2
SIZE ON THE ABA
MeABA:EtABA
EXTRACTED”
ratio
Sample FW (b9
(a)
(b)
Mean
Mean ABA content (ngig FW
0.10 0.15 0.20 0.25 0.30
0.62 0.62 0.72 0.66 0.61
0.70 0.64 0.73 0.71 0.69
0.66 0.63 0.73 0.69 0.65
71 68 79 74 70
a MeABA:EtABA
ratios were obtained with 2.92 ng of EtABA present.
data for sonication and elution efficiencies represent a mean recovery of MeABA from readily extractable ABA in the plant tissues of 81%. The reliability of the assay was found to depend largely upon the state of the ECD, which, because of the limited purification of each sample, would tend to become contaminated fairly readily. However, with carefully controlled operating conditions it was found that a detector could be used for about 1000 injections before its response became unreliable, necessitating cleaning of the radioactive source by the manufacturers. The process time for each leaf extract during continuous assaying was 40 min (20 min/assay), and the sensitivity of a clean detector was such that a tissue sample containing as little as 100 pg of ABA could be assayed reliably. This value represents a tissue fresh weight of less than a milligram for wheat leaves at a potential of - 12 to - 14 bar. For low tissue concentrations of ABA the sensitivity and accuracy of the assay were governed by the amount of material that could be applied to the thin-layer plate. In such cases it may be necessary to use a prepurification step to improve sensitivity. Although the assay was developed primarily to speed up routine determinations of ABA in water-stressed wheat leaves, the TABLE AMOUNTS
Initial extract MeABA:EtABA ratio
Resonicated extract MeABA:EtABA ratio
1
2
Mean
1.06 0.80 0.86
0.97 0.74 0.87
1.01
0.10
0.77 0.87
” MeABA:EtABA
3
OF ABA EXTRACTED INITIALLY FROM STRESSED LEAVES ON REEXTRACTION OF THE CENTRIFUGED RESIDUES”
AND
ABA (% of that initially extracted)
2
Mean
0.07
0.09 0.07
0.09 0.07
9 9
0.10
0.10
0.10
12
1
ratios were obtained with 2.92 ng of EtABA present.
155
RAPID ASSAY FOR ABSCISIC ACID
EtABA
,,,,,( *b, /
J
0
4 min.
a
0
4 rnln.
8
FIG. 6 (left). Assay of 3.5 mg of xylem sap collected from the cut surface ofa stressed wheat leaf under pressure in a pressure bomb; 1.17 ng of EtABA was added to the assay. EtABA peak represents l/20 of total EtABA present (58 pg). MeABA:EtABA ratio represents 205 ng of ABA/ml of xylem sap. FIG. 7 (right). Assay of 16 mg of lower epidermis removed from a stressed leaf of Vicinfuba; 1.17 ng of EtABA was added to the assay. MeABA:EtABA ratio represents 110 ng of ABA/g FW of epidermis.
sensitivity of the technique renders it ideal for studies of some of the fundamental questions concerning the physiological role of ABA in controlling stomata1 movements and other responses. For example, the ABA concentration in expressed xylem sap (Fig. 6) and in isolated leaf epidermis (Fig. 7) has been measured. ACKNOWLEDGMENT The expert technical assistance of Miss Pam Smith in the development of the assay and in its routine use is gratefully acknowledged.
156
S. A. QUARRJE
REFERENCES I. 2. 3. 4. 5. 6. 7. 8.
Wright, S. T. C. (1969) Planta 86, 10-20. Zabadal, T. J. (1974) Plant Physiol. 53, 125-127. Beardsell, M. F., and Cohen, D. (1975) Plant Physiol. 56, 207-212. Lenton, J. R., Perry, V. M., and Saunders, P. F. (1971) Planta 96, 271-280. Colquhoun, A. J., and Hillman, J. R. (1975) Z. PJanzenphysiol. 76, 326-332. Sweetser, P. B., and Vatvars, A. (1976) Anal. Biochem. 71, 68-78. Seeley, S. D., and Powell, L. E. (1970) Anal. Biochem. 35, 530-533. Mizrahi, Y., Blumenfeld, A., Bittner, S., and Richmond, A. E. (1971) Plant Physiol. 48, 752-755. 9. Loveys, B. R., and Kriedeman, P. E. (1974) Aust. .f. Planf Physiol. 1, 407-415. 10. Zeevaart, J. A. D. (1971) Plant Physiol. 48, 86-90. 11. Vogel, A. I. (1956) “Practical Organic Chemistry,” 3rd ed., p. 969. Longmans, London