Planta (Berl.) 123,291--298 (1975) 9 by Springer-Verlag 1975
The Abscisic Acid Content of Dormant Birch Buds* M. A. Harrison** and P. F. Saunders Department of Botany and Microbiology, The University College of Wales, Aberystwyth SY23 3DA, U.K. Received 23 December 1974; accepted 6 February 1975 Summary. Abscisic acid (ABA) has been identified in the buds and xylem sap of Betula verrucosa (Ehrh.). Buds also contain esterified ABA. In the course of the winter the proportion
of esterified ABA in the buds undergoes a progressive increase which may be associated with emergence from dormancy. Introduction
The idea t h a t the winter dormancy of temperate woody plants might be regulated by changes in the level of endogenous inhibitors was suggested by Hemberg (1949), who noted a gradual decline over winter in the inhibitory activity, measured by bioassay, of the acidic ether-soluble fraction of extracts of F r a x i n u s excelsior buds. Artificially breaking dormancy by applying 2-chloroethanol induced a similar change. Subsequently, much lower levels of "inhibitor fl" (Bennet-Clark and Kefford, 1953), were found in buds of F. excelsior in February, w h e n the buds were emerging from d o r m a n c y , t h a n in dormant buds harvested in October (Hemberg, 1958). Inhibitor fl levels in Aeer pseudoplatanus also declined over winter, reaching very low levels at the time of bud burst (Phillips and Wareing, 1958). Essentially similar findings have been reported in several other instances, levels of inhibitory substances declining to low levels or disappearing altogether with the approach of bud burst (Hendershott and Bailey, 1955; yon Guttenberg and Leike, 1958; E1 Antably, 1965 ; Kawase, 1966). Tinklin and Schwabe (1970) not only observed a rapid decline in inhibitor levels in lateral buds of Ribes nigra between December and February, but also showed that a similar decline could be obtained by chilling buds on shoots taken indoors at midwinter. Certain exceptions to the general picture of declining inhibitor levels before bud burst have, however, been reported. No decrease in the inhibitor content of peach buds was observed in one case until after bud break (Dennis and Edgerton, 1961), while yon Guttenberg and Leike (1958), although reporting a decline in buds of Aeer, were unable to detect a similar decline in Syrinffa. While bioassay data have frequently indicated t h a t inhibitor fl levels m a y fall in buds during the winter, the danger of assuming t h a t such changes reflect a decreased content of abscisie acid (ABA) has been clearly demonstrated in the case of dormancy induction by short days (Lenton et al (1972). I t is also significant to * Abbreviations: ABA, abscisic acid; PVP, polyvinyl pyrrolidone; GLC, gas-liquid chromatography; ORD, optical rotatory dispersion; TLC, Thin-layer chromatography. ** Present address: Department of Plant Biochemistry, State University College of Forestry, Syracuse, N.Y. 13210, USA
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n o t e in this c o n t e x t t h a t Corgan a n d P e y t o n (1970) h a v e r e p o r t e d t h a t , a l t h o u g h levels of i n h i b i t o r y a c t i v i t y in p e a c h flower buds decreased o v e r winter, t h e decline was m o r e m a r k e d in a " t o t a l e t h e r - s o l u b l e " f r a c t i o n t h a n in a " p u r i f i e d A B A " fraction. Clearly, t h e r e is an u r g e n t need to m e a s u r e seasonal v a r i a t i o n s in A B A c o n t e n t using direct m e t h o d s such as gas-liquid c h r o m a t o g r a p h y ( Len t o n et al., 1971). A c o n j u g a t e of A B A , t h e glucose ester, was first isolated f r o m fruit of L u p i n u s luteus b y K o s h i m i t z u et al. (1968), a n d was s u b s e q u e n t l y shown b y Milborrow (1970) to be a m a j o r p r o d u c t of th e m e t a b o l i s m of exogenous A B A b y t o m a t o shoots. I t could be suggested t h a t t h e esterification of A B A b y glucose m i g h t r e p r e s e n t a n a t u r a l m e c h a n i s m for controlling its i n h i b i t o r y a c t i v i t y . W i t h this possibility in mind, m e a s u r e m e n t s h a v e been m a d e of t h e a m o u n t s of free an d esterffied A B A in buds of B e t u l a verrucosa ( c o m m o n birch) collected from saplings a t i n t e r v a l s t h r o u g h o u t t h e w i n t e r of 1971-72. W i t h seedlings of Betula pubescens it has been shown t h a t l o n g - d a y t r e a t m e n t of leafless plants in t h e w a r m will p r o m o t e b u d b u r s t (Wareing, 1954). A period of chilling at 5 ~ C, however, accelerates b u d b u r s t e v e n in continuous darkness. I n t h e p r e s e n t work, some i n d i c a t i o n of t h e s t a t e of d o r m a n c y of buds on saplings was o b t a i n e d b y observing b u d break on twigs collected from saplings a t i n t e r v a l s a n d b r o u g h t into a g r o w t h cab i n et m a i n t a i n e d a t 20 ~ C u n d e r long d a y conditions.
Materials and Methods Plant Material. Bud and twig collections were made from a closely spaced row of 35 uniclonar grafted saplings of Betula verrucosa Ehrh., about 2 m in height, growing in the open in Aberystwyth. After harvest, buds were immediately frozen in liquid nitrogen while awaiting extraction. Extraction and Qualitative GLC. Buds were macerated at 5~ C in a blender using a volume in cma of chilled 80% aqueous methanol roughly equal to 15 times the sample fresh weight in grams. Sodium bicarbonate (5 g dm -3) was added to the homogcnates to maintain a neutral or slightly alkaline pH. All operations were carried out with minimal exposure to light. Homogenates were stirred for several hours in one half of the original volume of 80% methanol, filtered and washed ~wice with further one-eighth volumes of 80% methanol. The methanolic extracts were bulked and reduced to an aqueous solution on a rotary evaporator at 35~ C in vaeuo. The aqueous residue was frozen, thawed, adjusted if necessary to pH 7.0 and stirred for 1 h at 5~ C with 1 g insoluble polyvinylpyrrolidone (PVP) powder (Polyclar AT). PVP and detritus were removed by filtration, filtrates and washings were combined and the PVP treatment repeated if necessary. The resulting filtrate was adjusted to pH 3.5 and extracted three times with equal volumes of redistilled diethyl ether. Ether extracts were bulked, dried over anhydrous sodium sulphate and evaporated to dryness. The aqueous residue left after ether extraction was adjusted to pH 11.0 with potassium hydroxide solution and maintained for 1 h on a shaking water bath at 60~ C. After re-adjusting the pH to 3.5, the solution was extracted with ether as above to remove any ether-soluble materials released by alkaline hydrolysis. Thin-layer chromatography (TLC) of the purified ether-soluble fraction from the original extract and from the hydrolysate was carried out on 20 • 20 cm plates coated with a 0.5 mm layer of Merck Kieselgcl GF254 which had been slurried in a 0.3 % aqueous solution of carboxymethyl cellulose. Plates were washed by running in ethanol/acetic acid (98: 2; v/v) and activated at 100~ C for 30 rain. After loading, chromatograms were developed three times to a distance of 15 em in toluene/ethyl acetate/acetic acid (40:5:2; v/v/v). The plates were allowed to dry just sufficiently to allow location of ABA and 2trans-ABA marker spots under a UV lamp. The ABA zone was then immediately eluted with redistilled AR acetone.
ABA in Dormant Buds
293
After drying, eluates were dissolved in acetone/methanol (9:1 ; v/v) and methylated with diazomethane. For GLC, 1 mm 3 samples of the derivatised material, dissolved in acetone/ methanol (9:1; v/v), were ehromatographed at 210~ on a ttewlett-Paekard Model 402 chromatograph fitted with an electron capture detector. The stationary phase was Epon 1001 at a 2% loading on AW-DMCS Chromosorb W in 180 cm • 3 mm glass columns. Carrier gas was argon/methane (95:5) at a flow rate of 40 em3min-1. The identity of presumed methyl ABA peaks was confirmed by isomerisation under UV light (Lenton et al., 1971) or by spectropolarimetry using a Bellingham and Stanley Polarmatie sprectropolarimeter. Quantitative GLC. The foregoing methods were adequate for qualitative and rough quantitative work, although they involve an indeterminate loss of ABA during purification. For accurate quantitative analysis of ABA in original extracts or in hydrolysates, an isotope dilution technique was employed. A measured amount of [2-14C]ABA of known specific activity was added to the unfractionated extract which was then purified as described above. After elution from the thin layer plates a measured amount of 2,trans-ABA was added, measured aliquots were taken for radioactivity determinations using a Beckman LS 200 B scintillation spectrometer, and the remainder was methylated and used for GLC. From the relative areas of the ABA and 2,trans-ABA peaks on the gas ehromatogram, the total amount of ABA present in the TLC eluates was calculated. From the recovery of radioactivity, corrections could be applied for the amount of exogenous ABA in the purified sample and for loss of ABA up to and including the TLC stage. Results
Identi/ieation o/ A B A in Birch Buds and Spring Bleeding-Sap I n e a r l y e x p e r i m e n t s using birch buds, e x t r a c t s were p r e p a r e d a n d f r a c t i o n a t e d essentially as described b y L e n t o n et al. (1971). I n this m e t h o d , t h e zone from a t h i n l a y e r c h r o m a t o g r a m corresponding to t h e l~f of b o t h A B A a n d 2,trans-ABA is e l u t e d a n d used for d e r i v a t i s a t i o n a n d GLC. W i t h birch buds, GLC of such eluates i n d i c a t e d t h a t t h e e x t r a c t s contained a p p r e c i a b l e q u a n t i t i e s of m a t e r i a l corresponding in r e t e n t i o n t i m e to each of t h e two geometrical isomers. F o r example, t h e gas c h r o m a t o g r a m of a n e x t r a c t of buds h a r v e s t e d in late F e b r u a r y showed a p e a k corresponding to m e t h y l A B A a n d a second peak, a p p r o x i m a t e l y one t e n t h as large, corresponding to m e t h y l 2,trans-ABA. W h i l s t i t is possible t h a t 2,trans-ABA was f o r m e d as a n a r t i f a c t d u r i n g purification, g r e a t care was t a k e n to a v o i d conditions k n o w n to p r o m o t e isomerisation of A B A . I t seems probable, therefore, t h a t t h e second p e a k reflected t h e presence in t h e e x t r a c t either of 2,trans-ABA or of a c o m p o u n d h a v i n g a n identical r e t e n t i o n t i m e after d e r i v a t i s a tion. This p e a k has n o t been p o s i t i v e l y identified but, regardless of its n a t u r e , its presence m a d e it i m p r a c t i c a b l e to use t h e q u a n t i t a t i v e m e t h o d of L e n t o n et al. which depends on using a d d e d 2,trans-ABA as a n i n t e r n a l s t a n d a r d to correct for losses of A B A d u r i n g purification. Consequently, in t h e p r e s e n t work, TLC conditions were modified to p e r m i t complete s e p a r a t i o n of A B A a n d 2.trans-ABA, o n l y t h e A B A region of c h r o m a t o g r a m s was e l u t e d a n d 14C-labelled A B A was used as a n i n t e r n a l s t a n d a r d for q u a n t i t a t i v e analysis. A s a m p l e of buds h a r v e s t e d on 5 J a n u a r y 1972 was e x t r a c t e d a n d p r e p a r e d for q u a l i t a t i v e GLC as described u n d e r " M e t h o d s " . The gas c h r o m a t o g r a m of a s a m p l e d e r i v e d from t h e ether-soluble fraction a n d e q u i v a l e n t to 0.025 g fresh weight of b u d s is shown in Fig. 1 a. The large p e a k corresponded e x a c t l y in r e t e n t i o n t i m e with an a u t h e n t i c s a m p l e of m e t h y l A B A . Confirmation of t h e i d e n t i t y of this p e a k was o b t a i n e d b y placing a m e t h a n o l i c solution of t h e m e t h y l a t e d e x t r a c t in a silica c u v e t t e a n d s t a n d i n g for 5 h in t h e light from a I t a n o v i a Model 16 U V lamp.
294
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ABA
o~
2 trans ABA
,b
r
20
3'0
4J0
I
0
10 20 30 Retention time (rain)
4O
Fig. i a and b. Gas chromatogram of a methylated fraction purified from a birch bud extract (a) before, (b) after irradiation with UV light
After irradiation, a sample equivalent to 0.025 g fresh weight was rechromatographed giving the trace shown in Fig. 1 b. I t is apparent that about 50 % of the compound corresponding in retention time to methyl ABA was converted to a compound corresponding to methyl 2,trans-ABA. Similar behaviour was observed with a sample of authentic methyl ABA. The fraction remaining after ether extraction of the bud extract was divided into two equal portions. One portion was hydrolysed as described under "Methods". Both portions were then extracted at pH 3.5 with diethyl ether and the ether extracts purified for GLC. The resulting chromatograms indicated that approximately 35 times as much of a substance having a retention time identical with that of methyl ABA was present after hydrolysis than before hydrolysis. Confirmation of identity was obtained by isomerisation in UV light and re-chromatography which gave the expected approximately 1:1 ratio of peaks. On this basis it is concluded that birch buds contain appreciable quantities of esterified ABA. I n order to examine the ABA content of birch xylem sap, samples were collected of the exudate obtained from mature trees in early spring by boring into the sap wood. Again, initial experiments using the methods of Lenton et al. (loc. cir.) indicated the presence in methylated samples of a small but significant quantity of material having the same retention time as methyl 2,trans-ABA. Isotope dilution was, used for quantitative analysis of ABA content. With two samples, each comprising 250 cm 3 sap collected on 4 March 1972, quantitative GLC indicated the presence of 0.040 and 0.043 lzg ABA per cm 3 of sap respectively. Since the samples were very much cleaner than those obtained from buds, it proved possible to confirm the identity of the presumptive ABA using spectropolarimetry. A 500 cm 3 sample from the 4 March collection was purified in the usual way and, after TLC, was dissolved in 1 cm a of 0.005 tool dm -3 suIphuric acid and examined in a spectropolarimeter. The ORD curve obtained agreed well in all respects with published
ABA in Dormant Buds
295
@ I I
~1o
.J" LC
io
0
T
Sept
]
Oct
I
Nov
I
Dec
I
Jan
I
Feb
Mar
Fig. 2. Mean fresh weight of birch buds harvested at monthly intervals
data (Milborrow, 1967). Using the value given by Milborrow for the specific rotation of the naturally occurring enantiomer of ABA, the ORD measurements indicated the presence of 0.034 ~g ABA per cm a of sap. Assuming a recovery during purification of about 75 %, which is typical, this figure agrees well with the values obtained by isotope dilution. Examination of an all, aline hydrolysate of the aqueous fraction remaining after purifying free ABA from a xylem sap sample failed to demonstrate the presence of significant amounts of esterified ABA in birch spring sap.
Seasonal Variations in the A B A Content o/Birch Buds To facilitate collection of buds, clonM birch saplings rather than mature trees were used in a study of seasonal variations in the content of free and esterified ABA. In this material, leaf fall occurred in mid-November and at about the same time a marked drop was seen in the rate of increase of bud fresh weight (Fig. 2). Samples containing several thousand buds were obtained at monthly intervals between September 1971 and March 1972 by removing all undamaged vegetative buds on first year shoots from groups of three to five saplings. At each bud harvest, samples of ten twigs were excised immediately above the 1970 girdle scar. These were stood in water in a growth cabinet maintained at 20 ~ C with 16 h photoperiods. Water was changed once a week and the bottom few millimetres of each twig was excised. Bud break was recorded as having taken place on a twig when at least one bud had swollen and burst to the stage where ieaf unrolling was just beginning. The time taken for bud break to occur on each twig in successive harvests is shown in Fig. 3. Samples consisting of 10 g fresh weight of buds were taken from each monthly harvest and the content of free and esterified ABA was determined by quantitative GLC using isotope dilution. The data are presented in Table 1. Replicate samples were analysed for the September harvest and the values obtained are in good agreement. Lack of plant material precluded further replication but our experience with the technique allows us to place confidence in the significance of differences greater than about 5 %. The apparently random variations seen between treatments are, therefore, almost certainly real. Moreover, measurement of the mean fresh
296
)5. A. Harrison and P. F. Saunders
3oF/
0 10 0 10 0 10 0 10 0 10 0 10 0 10 Mar Sept Oct Nov Dec Jan F~b :Fig. 3. Time taken for bud break to occur on each of ten twigs in samples harvested at monthly intervals and maintained at 20 ~ C under long day conditions
weight of buds in successive harvests indicate that the variations are not a consequence of expressing ABA levels on a fresh weight basis.
Discussion From Fig. 3 it is apparent that any absolute requirement for chilling to break dormancy in the buds had been satisfied by mid-November at the latest. Thereafter bud break under favourable conditions became increasingly more rapid. With these observations in mind, it would be tempting to speculate that the progressive reduction in the time taken for buds to burst on excised twigs reflects a gradual reduction in their content of ABA at the time of harvest. From Table 1, however, it is clear that ABA levels fluctuate quite widely from month to month although there is an overall reduction of about 75% from September to March. For example, the content of free ABA in J a n u a r y is considerably greater than in December and only very slightly less than in October. A complicating factor in interpreting these measurements is the fact that the xylem sap of several trees has been shown to contain ABA (Lenton et al., 1968). In the course of the present work, sap collected in early March was found to contain 0.04 ~g ABA per cm a and, by analogy with willow, levels could be expected to be much higher in earlier months (Alvim and Saunders, unpublished). I t is reasonable to suppose that this ABA will reach the buds as the sap rises and that the buds are somehow protected from inhibition by it when growth commences in the spring. Conjugation with glucose could possibly afford such protection and, on this basis, it could be suggested that emergence from winter dormancy is associated with an increased capacity to conjugate ABA. The data presented in Table 1 support such a hypothesis. From October to February, levels of free and esterified ABA fluctuate in a parallel fashion. Between February and March, however, a large increase in the total ABA content can be entirely accounted for by an increase in esterified ABA while the amount of free ABA in the buds shows, if anything, a slight decrease. I t is suggested that this increase in esterified ABA represents conjugation of the free acid reaching the buds via the xylem sap. The relative pool sizes in the bud tissue of free and esterified ABA will clearly dependent on m a n y factors, including the rate of conversion of ABA to other
ABA in Dormant Buds
297
Table 1. The levels of free and esterified ABA in birch buds collected at monthly intervals Time of harvest
ABA content qxg/g fresh weight)
September 15th September 15th October 15th November 15th December 15th January 15th February 14th March 14th
Free
Esterified
1.17 1.21 0.86 0.96 0.52 0.76 0.31 0.27
0.35 0.33 0.74 1.08 0.65 1.08 0.69 1.85
10C 9(3 8O -o
40
o
"~ 70
30 ~50
t0 o
2O
The Abscisic Acid Content of Dormant Birch Buds* M. A. Harrison** and P. F. Saunders Department of Botany and Microbiology, The University College of Wales, Aberystwyth SY23 3DA, U.K. Received 23 December 1974; accepted 6 February 1975 Summary. Abscisic acid (ABA) has been identified in the buds and xylem sap of Betula verrucosa (Ehrh.). Buds also contain esterified ABA. In the course of the winter the proportion
of esterified ABA in the buds undergoes a progressive increase which may be associated with emergence from dormancy. Introduction
The idea t h a t the winter dormancy of temperate woody plants might be regulated by changes in the level of endogenous inhibitors was suggested by Hemberg (1949), who noted a gradual decline over winter in the inhibitory activity, measured by bioassay, of the acidic ether-soluble fraction of extracts of F r a x i n u s excelsior buds. Artificially breaking dormancy by applying 2-chloroethanol induced a similar change. Subsequently, much lower levels of "inhibitor fl" (Bennet-Clark and Kefford, 1953), were found in buds of F. excelsior in February, w h e n the buds were emerging from d o r m a n c y , t h a n in dormant buds harvested in October (Hemberg, 1958). Inhibitor fl levels in Aeer pseudoplatanus also declined over winter, reaching very low levels at the time of bud burst (Phillips and Wareing, 1958). Essentially similar findings have been reported in several other instances, levels of inhibitory substances declining to low levels or disappearing altogether with the approach of bud burst (Hendershott and Bailey, 1955; yon Guttenberg and Leike, 1958; E1 Antably, 1965 ; Kawase, 1966). Tinklin and Schwabe (1970) not only observed a rapid decline in inhibitor levels in lateral buds of Ribes nigra between December and February, but also showed that a similar decline could be obtained by chilling buds on shoots taken indoors at midwinter. Certain exceptions to the general picture of declining inhibitor levels before bud burst have, however, been reported. No decrease in the inhibitor content of peach buds was observed in one case until after bud break (Dennis and Edgerton, 1961), while yon Guttenberg and Leike (1958), although reporting a decline in buds of Aeer, were unable to detect a similar decline in Syrinffa. While bioassay data have frequently indicated t h a t inhibitor fl levels m a y fall in buds during the winter, the danger of assuming t h a t such changes reflect a decreased content of abscisie acid (ABA) has been clearly demonstrated in the case of dormancy induction by short days (Lenton et al (1972). I t is also significant to * Abbreviations: ABA, abscisic acid; PVP, polyvinyl pyrrolidone; GLC, gas-liquid chromatography; ORD, optical rotatory dispersion; TLC, Thin-layer chromatography. ** Present address: Department of Plant Biochemistry, State University College of Forestry, Syracuse, N.Y. 13210, USA
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M.A. Harrison and P. F. Saunders
n o t e in this c o n t e x t t h a t Corgan a n d P e y t o n (1970) h a v e r e p o r t e d t h a t , a l t h o u g h levels of i n h i b i t o r y a c t i v i t y in p e a c h flower buds decreased o v e r winter, t h e decline was m o r e m a r k e d in a " t o t a l e t h e r - s o l u b l e " f r a c t i o n t h a n in a " p u r i f i e d A B A " fraction. Clearly, t h e r e is an u r g e n t need to m e a s u r e seasonal v a r i a t i o n s in A B A c o n t e n t using direct m e t h o d s such as gas-liquid c h r o m a t o g r a p h y ( Len t o n et al., 1971). A c o n j u g a t e of A B A , t h e glucose ester, was first isolated f r o m fruit of L u p i n u s luteus b y K o s h i m i t z u et al. (1968), a n d was s u b s e q u e n t l y shown b y Milborrow (1970) to be a m a j o r p r o d u c t of th e m e t a b o l i s m of exogenous A B A b y t o m a t o shoots. I t could be suggested t h a t t h e esterification of A B A b y glucose m i g h t r e p r e s e n t a n a t u r a l m e c h a n i s m for controlling its i n h i b i t o r y a c t i v i t y . W i t h this possibility in mind, m e a s u r e m e n t s h a v e been m a d e of t h e a m o u n t s of free an d esterffied A B A in buds of B e t u l a verrucosa ( c o m m o n birch) collected from saplings a t i n t e r v a l s t h r o u g h o u t t h e w i n t e r of 1971-72. W i t h seedlings of Betula pubescens it has been shown t h a t l o n g - d a y t r e a t m e n t of leafless plants in t h e w a r m will p r o m o t e b u d b u r s t (Wareing, 1954). A period of chilling at 5 ~ C, however, accelerates b u d b u r s t e v e n in continuous darkness. I n t h e p r e s e n t work, some i n d i c a t i o n of t h e s t a t e of d o r m a n c y of buds on saplings was o b t a i n e d b y observing b u d break on twigs collected from saplings a t i n t e r v a l s a n d b r o u g h t into a g r o w t h cab i n et m a i n t a i n e d a t 20 ~ C u n d e r long d a y conditions.
Materials and Methods Plant Material. Bud and twig collections were made from a closely spaced row of 35 uniclonar grafted saplings of Betula verrucosa Ehrh., about 2 m in height, growing in the open in Aberystwyth. After harvest, buds were immediately frozen in liquid nitrogen while awaiting extraction. Extraction and Qualitative GLC. Buds were macerated at 5~ C in a blender using a volume in cma of chilled 80% aqueous methanol roughly equal to 15 times the sample fresh weight in grams. Sodium bicarbonate (5 g dm -3) was added to the homogcnates to maintain a neutral or slightly alkaline pH. All operations were carried out with minimal exposure to light. Homogenates were stirred for several hours in one half of the original volume of 80% methanol, filtered and washed ~wice with further one-eighth volumes of 80% methanol. The methanolic extracts were bulked and reduced to an aqueous solution on a rotary evaporator at 35~ C in vaeuo. The aqueous residue was frozen, thawed, adjusted if necessary to pH 7.0 and stirred for 1 h at 5~ C with 1 g insoluble polyvinylpyrrolidone (PVP) powder (Polyclar AT). PVP and detritus were removed by filtration, filtrates and washings were combined and the PVP treatment repeated if necessary. The resulting filtrate was adjusted to pH 3.5 and extracted three times with equal volumes of redistilled diethyl ether. Ether extracts were bulked, dried over anhydrous sodium sulphate and evaporated to dryness. The aqueous residue left after ether extraction was adjusted to pH 11.0 with potassium hydroxide solution and maintained for 1 h on a shaking water bath at 60~ C. After re-adjusting the pH to 3.5, the solution was extracted with ether as above to remove any ether-soluble materials released by alkaline hydrolysis. Thin-layer chromatography (TLC) of the purified ether-soluble fraction from the original extract and from the hydrolysate was carried out on 20 • 20 cm plates coated with a 0.5 mm layer of Merck Kieselgcl GF254 which had been slurried in a 0.3 % aqueous solution of carboxymethyl cellulose. Plates were washed by running in ethanol/acetic acid (98: 2; v/v) and activated at 100~ C for 30 rain. After loading, chromatograms were developed three times to a distance of 15 em in toluene/ethyl acetate/acetic acid (40:5:2; v/v/v). The plates were allowed to dry just sufficiently to allow location of ABA and 2trans-ABA marker spots under a UV lamp. The ABA zone was then immediately eluted with redistilled AR acetone.
ABA in Dormant Buds
293
After drying, eluates were dissolved in acetone/methanol (9:1 ; v/v) and methylated with diazomethane. For GLC, 1 mm 3 samples of the derivatised material, dissolved in acetone/ methanol (9:1; v/v), were ehromatographed at 210~ on a ttewlett-Paekard Model 402 chromatograph fitted with an electron capture detector. The stationary phase was Epon 1001 at a 2% loading on AW-DMCS Chromosorb W in 180 cm • 3 mm glass columns. Carrier gas was argon/methane (95:5) at a flow rate of 40 em3min-1. The identity of presumed methyl ABA peaks was confirmed by isomerisation under UV light (Lenton et al., 1971) or by spectropolarimetry using a Bellingham and Stanley Polarmatie sprectropolarimeter. Quantitative GLC. The foregoing methods were adequate for qualitative and rough quantitative work, although they involve an indeterminate loss of ABA during purification. For accurate quantitative analysis of ABA in original extracts or in hydrolysates, an isotope dilution technique was employed. A measured amount of [2-14C]ABA of known specific activity was added to the unfractionated extract which was then purified as described above. After elution from the thin layer plates a measured amount of 2,trans-ABA was added, measured aliquots were taken for radioactivity determinations using a Beckman LS 200 B scintillation spectrometer, and the remainder was methylated and used for GLC. From the relative areas of the ABA and 2,trans-ABA peaks on the gas ehromatogram, the total amount of ABA present in the TLC eluates was calculated. From the recovery of radioactivity, corrections could be applied for the amount of exogenous ABA in the purified sample and for loss of ABA up to and including the TLC stage. Results
Identi/ieation o/ A B A in Birch Buds and Spring Bleeding-Sap I n e a r l y e x p e r i m e n t s using birch buds, e x t r a c t s were p r e p a r e d a n d f r a c t i o n a t e d essentially as described b y L e n t o n et al. (1971). I n this m e t h o d , t h e zone from a t h i n l a y e r c h r o m a t o g r a m corresponding to t h e l~f of b o t h A B A a n d 2,trans-ABA is e l u t e d a n d used for d e r i v a t i s a t i o n a n d GLC. W i t h birch buds, GLC of such eluates i n d i c a t e d t h a t t h e e x t r a c t s contained a p p r e c i a b l e q u a n t i t i e s of m a t e r i a l corresponding in r e t e n t i o n t i m e to each of t h e two geometrical isomers. F o r example, t h e gas c h r o m a t o g r a m of a n e x t r a c t of buds h a r v e s t e d in late F e b r u a r y showed a p e a k corresponding to m e t h y l A B A a n d a second peak, a p p r o x i m a t e l y one t e n t h as large, corresponding to m e t h y l 2,trans-ABA. W h i l s t i t is possible t h a t 2,trans-ABA was f o r m e d as a n a r t i f a c t d u r i n g purification, g r e a t care was t a k e n to a v o i d conditions k n o w n to p r o m o t e isomerisation of A B A . I t seems probable, therefore, t h a t t h e second p e a k reflected t h e presence in t h e e x t r a c t either of 2,trans-ABA or of a c o m p o u n d h a v i n g a n identical r e t e n t i o n t i m e after d e r i v a t i s a tion. This p e a k has n o t been p o s i t i v e l y identified but, regardless of its n a t u r e , its presence m a d e it i m p r a c t i c a b l e to use t h e q u a n t i t a t i v e m e t h o d of L e n t o n et al. which depends on using a d d e d 2,trans-ABA as a n i n t e r n a l s t a n d a r d to correct for losses of A B A d u r i n g purification. Consequently, in t h e p r e s e n t work, TLC conditions were modified to p e r m i t complete s e p a r a t i o n of A B A a n d 2.trans-ABA, o n l y t h e A B A region of c h r o m a t o g r a m s was e l u t e d a n d 14C-labelled A B A was used as a n i n t e r n a l s t a n d a r d for q u a n t i t a t i v e analysis. A s a m p l e of buds h a r v e s t e d on 5 J a n u a r y 1972 was e x t r a c t e d a n d p r e p a r e d for q u a l i t a t i v e GLC as described u n d e r " M e t h o d s " . The gas c h r o m a t o g r a m of a s a m p l e d e r i v e d from t h e ether-soluble fraction a n d e q u i v a l e n t to 0.025 g fresh weight of b u d s is shown in Fig. 1 a. The large p e a k corresponded e x a c t l y in r e t e n t i o n t i m e with an a u t h e n t i c s a m p l e of m e t h y l A B A . Confirmation of t h e i d e n t i t y of this p e a k was o b t a i n e d b y placing a m e t h a n o l i c solution of t h e m e t h y l a t e d e x t r a c t in a silica c u v e t t e a n d s t a n d i n g for 5 h in t h e light from a I t a n o v i a Model 16 U V lamp.
294
)r A. Harrison and P. F. Saunders
ABA
o~
2 trans ABA
,b
r
20
3'0
4J0
I
0
10 20 30 Retention time (rain)
4O
Fig. i a and b. Gas chromatogram of a methylated fraction purified from a birch bud extract (a) before, (b) after irradiation with UV light
After irradiation, a sample equivalent to 0.025 g fresh weight was rechromatographed giving the trace shown in Fig. 1 b. I t is apparent that about 50 % of the compound corresponding in retention time to methyl ABA was converted to a compound corresponding to methyl 2,trans-ABA. Similar behaviour was observed with a sample of authentic methyl ABA. The fraction remaining after ether extraction of the bud extract was divided into two equal portions. One portion was hydrolysed as described under "Methods". Both portions were then extracted at pH 3.5 with diethyl ether and the ether extracts purified for GLC. The resulting chromatograms indicated that approximately 35 times as much of a substance having a retention time identical with that of methyl ABA was present after hydrolysis than before hydrolysis. Confirmation of identity was obtained by isomerisation in UV light and re-chromatography which gave the expected approximately 1:1 ratio of peaks. On this basis it is concluded that birch buds contain appreciable quantities of esterified ABA. I n order to examine the ABA content of birch xylem sap, samples were collected of the exudate obtained from mature trees in early spring by boring into the sap wood. Again, initial experiments using the methods of Lenton et al. (loc. cir.) indicated the presence in methylated samples of a small but significant quantity of material having the same retention time as methyl 2,trans-ABA. Isotope dilution was, used for quantitative analysis of ABA content. With two samples, each comprising 250 cm 3 sap collected on 4 March 1972, quantitative GLC indicated the presence of 0.040 and 0.043 lzg ABA per cm 3 of sap respectively. Since the samples were very much cleaner than those obtained from buds, it proved possible to confirm the identity of the presumptive ABA using spectropolarimetry. A 500 cm 3 sample from the 4 March collection was purified in the usual way and, after TLC, was dissolved in 1 cm a of 0.005 tool dm -3 suIphuric acid and examined in a spectropolarimeter. The ORD curve obtained agreed well in all respects with published
ABA in Dormant Buds
295
@ I I
~1o
.J" LC
io
0
T
Sept
]
Oct
I
Nov
I
Dec
I
Jan
I
Feb
Mar
Fig. 2. Mean fresh weight of birch buds harvested at monthly intervals
data (Milborrow, 1967). Using the value given by Milborrow for the specific rotation of the naturally occurring enantiomer of ABA, the ORD measurements indicated the presence of 0.034 ~g ABA per cm a of sap. Assuming a recovery during purification of about 75 %, which is typical, this figure agrees well with the values obtained by isotope dilution. Examination of an all, aline hydrolysate of the aqueous fraction remaining after purifying free ABA from a xylem sap sample failed to demonstrate the presence of significant amounts of esterified ABA in birch spring sap.
Seasonal Variations in the A B A Content o/Birch Buds To facilitate collection of buds, clonM birch saplings rather than mature trees were used in a study of seasonal variations in the content of free and esterified ABA. In this material, leaf fall occurred in mid-November and at about the same time a marked drop was seen in the rate of increase of bud fresh weight (Fig. 2). Samples containing several thousand buds were obtained at monthly intervals between September 1971 and March 1972 by removing all undamaged vegetative buds on first year shoots from groups of three to five saplings. At each bud harvest, samples of ten twigs were excised immediately above the 1970 girdle scar. These were stood in water in a growth cabinet maintained at 20 ~ C with 16 h photoperiods. Water was changed once a week and the bottom few millimetres of each twig was excised. Bud break was recorded as having taken place on a twig when at least one bud had swollen and burst to the stage where ieaf unrolling was just beginning. The time taken for bud break to occur on each twig in successive harvests is shown in Fig. 3. Samples consisting of 10 g fresh weight of buds were taken from each monthly harvest and the content of free and esterified ABA was determined by quantitative GLC using isotope dilution. The data are presented in Table 1. Replicate samples were analysed for the September harvest and the values obtained are in good agreement. Lack of plant material precluded further replication but our experience with the technique allows us to place confidence in the significance of differences greater than about 5 %. The apparently random variations seen between treatments are, therefore, almost certainly real. Moreover, measurement of the mean fresh
296
)5. A. Harrison and P. F. Saunders
3oF/
0 10 0 10 0 10 0 10 0 10 0 10 0 10 Mar Sept Oct Nov Dec Jan F~b :Fig. 3. Time taken for bud break to occur on each of ten twigs in samples harvested at monthly intervals and maintained at 20 ~ C under long day conditions
weight of buds in successive harvests indicate that the variations are not a consequence of expressing ABA levels on a fresh weight basis.
Discussion From Fig. 3 it is apparent that any absolute requirement for chilling to break dormancy in the buds had been satisfied by mid-November at the latest. Thereafter bud break under favourable conditions became increasingly more rapid. With these observations in mind, it would be tempting to speculate that the progressive reduction in the time taken for buds to burst on excised twigs reflects a gradual reduction in their content of ABA at the time of harvest. From Table 1, however, it is clear that ABA levels fluctuate quite widely from month to month although there is an overall reduction of about 75% from September to March. For example, the content of free ABA in J a n u a r y is considerably greater than in December and only very slightly less than in October. A complicating factor in interpreting these measurements is the fact that the xylem sap of several trees has been shown to contain ABA (Lenton et al., 1968). In the course of the present work, sap collected in early March was found to contain 0.04 ~g ABA per cm a and, by analogy with willow, levels could be expected to be much higher in earlier months (Alvim and Saunders, unpublished). I t is reasonable to suppose that this ABA will reach the buds as the sap rises and that the buds are somehow protected from inhibition by it when growth commences in the spring. Conjugation with glucose could possibly afford such protection and, on this basis, it could be suggested that emergence from winter dormancy is associated with an increased capacity to conjugate ABA. The data presented in Table 1 support such a hypothesis. From October to February, levels of free and esterified ABA fluctuate in a parallel fashion. Between February and March, however, a large increase in the total ABA content can be entirely accounted for by an increase in esterified ABA while the amount of free ABA in the buds shows, if anything, a slight decrease. I t is suggested that this increase in esterified ABA represents conjugation of the free acid reaching the buds via the xylem sap. The relative pool sizes in the bud tissue of free and esterified ABA will clearly dependent on m a n y factors, including the rate of conversion of ABA to other
ABA in Dormant Buds
297
Table 1. The levels of free and esterified ABA in birch buds collected at monthly intervals Time of harvest
ABA content qxg/g fresh weight)
September 15th September 15th October 15th November 15th December 15th January 15th February 14th March 14th
Free
Esterified
1.17 1.21 0.86 0.96 0.52 0.76 0.31 0.27
0.35 0.33 0.74 1.08 0.65 1.08 0.69 1.85
10C 9(3 8O -o
40
o
"~ 70
30 ~50
t0 o
2O
