Planta (Berl.) 94, 213--219 (1970) 9 by Springer-Verlag 1970

Identification of Abscisic Acid in Flower Buds of Coffea arabica (L.) G. B R O W ~ G and G. V. HOAD Long Ashton Research Station, University of Bristol P. GASKlYN Department of Organic Chemistry, University of Bristol Received June 30, 1970 Summary. Extracts of flower buds of Co[/ea arabica (L.) collected before and after bud break contain abscisic acid. This was demonstrated using thin layer chromatography and gas chromatography combined with mass spectroscopy. Abscisie acid accounts for about 75 % of the inhibitory activity in the acidic extract. The possible role of abseisic acid in the dormancy of coffee flower buds is discussed.

Indroduction I n the field flower buds of Co/lea arabica (L.) stop growing when 4-6 m m long and remain dormant until exposed to rain or irrigation, 8-15 days after which the flowers open. The factors responsible for coffee flower buds becoming dormant are not understood. Using controlled environments, W e n t (1957) found t h a t flower bud dormancy was more marked at high temperatures, and if trees were grown continuously under a 23 ~ C day/17 ~ C night regime flowers opened in flushes without other treatments. F r o m similar experiments, Mes (1957) concluded t h a t dorm a n c y could not be induced b y temperature per se as buds remained dorm a n t for a time even under a 20 ~ C day/14 ~ C night regime. Clearly, however, dormancy can be modulated b y temperature. Mes (1957) has also proposed t h a t dormancy is induced by a Iocalised water deficit in the flower buds, but this hypothesis has to be considered in relation to the observations of other workers, t h a t prior exposure to water stress is necessary before flower bud dormancy can be broken (Alvim, 1960; Rees, 1964; v a n der Veen, 1968). Several other investigations have indicated t h a t hormones m a y be involved in the mechanisms controlling dormancy and break in coffee flower buds. Thus, bud break can be induced by spraying flower buds with aqueous solutions of gibberellie acid (GA) (Alvim, 1958; Pagaez, 1959) and b y applying GA in lanolin (van der Veen, 1968) or in microdrops of ethanol directly to buds (Browning, unpublished). Alvim (1958)

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suggested t h a t t h e gibberellin levels in coffee trees m i g h t increase after exposure t o r a i n or irrigation. Using Col/ca canephora var. robusta growing in t h e greenhouse, v a n der Veen (1968) could n o t o b t a i n a n y response to t h e c y t o k i u i n k i n e t i n a p p l i e d in lanolin to d o r m a n t buds. H o w e v e r , a p p l y i n g t h e s y n t h e t i c c y t o k i n i n b e n z y l a d e n i n e (BA) in m i c r o - d r o p s of e t h a n o l to b u d s of Col]ca arabica (L.) growing in t h e field does b r e a k d o r m a n c y if t h e trees h a v e been s u b j e c t e d to d r o u g h t for one m o n t h (Browning, unpublished). A t t e m p t s to b r e a k d o r m a n c y w i t h auxins h a v e been unsuccessful (Mes, 1957; W o r m e r , 1965). V a n der Veen (1968) w o r k i n g w i t h Co/lea canephora var. robusta f o u n d t h a t 200~g of abseisic a c i d (ABA) a p p l i e d in lanolin t o d o r m a n t b u d s p r e v e n t e d b u d b r e a k a n d suggested t h a t endogenous A B A m i g h t also impose d o r m a n c y . I n a n a t t e m p t to e x p l a i n t h e w a t e r deficit effect on b u d d o r m a n c y A l v i m (1960) also i n v o k e d a n i n h i b i t o r as t h e cause of d o r m a n c y . A B A has been identified in e x t r a c t s f r o m m a n y p l a n t s (Milborrow, 1967) a n d is t h o u g h t to p l a y a p a r t in t h e w i n t e r d o r m a n c y of t e m p e r a t e trees (Eagles a n d W a r e i n g , 1964; Cornforth et al., 1965; Bowen a n d H o a d , 1967). H o w e v e r , A B A has n o t so far been identified in b u d e x t r a c t s from a t r o p i c a l species, n o r h a s i t been identified in flower b u d e x t r a c t s from a n y species. I n t h e p r e s e n t work, e x t r a c t s of coffee flower b u d s collected before a n d after b u d b r e a k were f o u n d to c o n t a i n a n acidic i n h i b i t o r y substance, which has been i d e n t i f i e d as abseisic acid.

Materials and Methods Flower buds were collected from six year old trees of Col/ca arabica (L.), cvs. SL. 34 and 28, growing in the field at Ruiru, Kenya. The samples were frozen on dry ice, lyophilised, milled to a powder, and then air-freighted to Long Ashton Research Station, England, where they were deep frozen until extracted. The material extracted included buds collected on March 24th and 26th 1969, follo~ving rain-induced bud break on March 24th, and dormant buds collected on April 14th and 17th. The lyophilised powder (100 or 200 g) was saturated with phosphate buffer at pH 4.0 (1 ml per g) and extracted for 16 hours at 3~ C with methylene dichloride (5 ml per g). After filtering, the residue was washed with a further 1 ml per g of solvent (after Luckwill et aL, 1969). The combined methylene dichloride extracts were taken to dryness under reduced pressure and redissolved in 100 ml methylene dichloride. This was partitioned three times against half volumes of 0.1 M phosphate buffer at pH 8.0, and the combined aqueous phases partitioned twice against equal volumes of petroleum ether, Iollowed by five half volumes of ethyl acetate. The aqueous extract was adjusted to pH 3.0 with 2N HC1, and partitioned a further five times with half volumes of ethyl acetate. After evaporation under reduced pressure, the acidic ethyl acetate extract was eluted with I M formic acid from a 28 • 2era column of Dowex I (50-100 mesh) formate form ion exchange column. The formic acid eluate was taken to dryness, placed on a 28 • 2 cm column of charcoal (Darco G-50) : Celite 535 (1:2) and einted with increasing proportions of acetone in water in 10% steps (Ohkuma et al., 1963). All solvents used were redistilled before use.

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Paper chromatography was done on solvent-washed strips of Whatman No. 3 paper, 5 cm wide, on to which extracts were line-loaded and the solvent allowed to run 20 cm in the descending direction. After development, the chromatograms were cut into 10 strips corresponding to 0.1 Rf values. These were eluted in 4.5 cm diameter petri dishes with 1 ml buffered sucrose, and then bioassayed. Thin layer chromatography was done on solvent washed 300 m9 layers of silica gel GF 254 (Merck) spread on 20 • 20 cm plates. Extracts and abscisic acid markers were applied to the plates in the manner described by Milborrow (1967). After development, the plates were examined under a UV lamp and UV absorbing bands marked. The silica gel was scraped from the plate in bands corresponding to 0.1 R f values and eluted with water saturated ethyl acetate. Small volumes of the elutant were dried on 4.25 cm Whatman No. 1 filter paper discs in 4.5 cm diameter petri dishes, eluted with 1 ml buffered sucrose, and then bioassayed. Preliminary gas chromatography of the samples was performed on a Varian Aerograph 205B using a 5 f t • 1/8 in. glass column of 2% QF1 on silanized "Gaschrom A". The temperature of the oven was kept constant at 170 ~ C or was programmed between 148 ~ and 200 ~ C at 6~ after an initial hold of 8 min at 148 ~ C. The injector and detector temperatures used were 210 ~ and 237 ~ respectively. For combined gas chromatography-mass spectroscopy (G. C.-M. S.) gas chromatography was performed on a Varian Aerograph 1200 using a 10 f t • 1/16 in. glass column of 2 % SE 33 on 100-120 mesh Gaschrom Q. The oven temperature was programmed from 210 ~ to 230 ~ C at 2~ with an injector temperature of 240 ~ C. Peaks in the total ion current trace were scanned using a Varian MAT CH-7 mass spectrometer. Coffee bud extracts and authentic ABA were methylated using ethereal diazomethane. Biological activity was determined using ten 10 mm long sub-apical sections of wheat coleoptfles cv. Atle, incubated in the dark for 20 hours at 25 ~ C. Responses at different R r values were tested for statistical significance at P < 0.01. For quantitative bioassay, extracts at four geometric dilutions and ABA at five geometric dilutions were tested. After logarithmic transformations the dose/response curves obtained were tested for linearity and parallelism using regression analysis, and the concentration of inhibiter in the extract estimated with confidence limits at P < 0.05 by a parallel-line method. For comparison with the estimates by bioassay of ABA in extracts, a further estimation was made by Optical Rotatory Dispersion using a Bellingham and Stanley Polarmatic 62 Spectropolarimeter.

Results and Discussion T h e acidic e t h y l a c e t a t e e x t r a c t w a s f o u n d b y b i o a s s a y t o c o n t a i n a n i n h i b i t o r r u n n i n g a t R f v a l u e s 0.6 t o 0.8 a n d 0.7 t o 0.9 on p a p e r c h r o m a t e g r a m s d e v e l o p e d in I s o p r o p a n o l : a m m o n i a : w a t e r (1O: 1:1) a n d Isopropanol: water: ammonia (80:19.95:0.05) respectively. These Rf v a l u e s c o r r e s p o n d t o t h o s e of a u t h e n t i c A B A i n t h e s a m e s o l v e n t s y s t e m s . N o i n h i b i t o r y a c t i v i t y a t t h e s e R f v a l u e s was f o u n d in t h e n e u t r a l e t h y l acetate extract. T h e acidic e t h y l a c e t a t e e x t r a c t i o n y i e l d e d a p p r o x i m a t e l y 7 5 0 ~ g / g extracted.

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Further purification of this extract by elution in 1.0 M formic acid from a Dowex I formate form ion-exchange column reduced the weight to approximately 90 ~g/g extracted. This procedure removed the viscous, coloured material in the extract, which was found to bind irreversibly to the resin. Approximately 85 % of the inhibitory activity was recovered in the formic acid. Authentic ABA could be recovered in 80% yield from an identical column. The extract was then chromatogrammed on a charcoal: celite (1:2) column and the inhibitor recovered in 60 and 70 % acetone in water. The 60 and 70 % fractions were combined giving a weight of about 13 ~g/g extracted and were run on thin layer plates developed in n-propanol :n-butanol :ammonia : water (6 : 2 : 1 : 2). A band showing UV absorbance and inhibitory activity at the same Rf values as the ABA markers (see Table) was run again on thin layer using ethyl acetate as the solvent. The UV absorbing and inhibitory band again co-chromatographed with the ABA markers (see Table) and, after elution, was used as the final inhibitor extract. This weighed approximately 11~g/g extracted. The coffee bud inhibitor was found also to cochromatograph with authentic ABA on TLC using three other solvent systems, as shown in the Table.

Table. R I values on TLC using GF/254 /or the coffee /lower bud inhibitor and authentic A B A in five di//erent solvent systems Solvent system

Rf values coffee bud inhibitor

ABA

1 2 3 4 5

0.50--0.56 0.26--0.32 0.40--0.45 0.57--0.64 0.83--0.89

0.53 0.29 0.43 0.60 0.86

1. n propanol; n butanol; ammonia; water (6:2:1:2) 2. Ethyl acetate 3. Benzene; ethyl acetate; acetic acid (50:100: 2) 4. Isopropanol; ammonia; water (10:1:1) 5. Water

Further evidence for the identity of the coffee bud inhibitor with abscisic acid was obtained, when after methylation with diazomethane and examination by gas chromatography using QF1 columns, the final inhibitor extract was found to contain a methyl ester with the same retention

Abscisic Acid in Flower Buds

I

t

+ +

i

i

i

I

i

217

~

Time rain

Fig. 1. G. C. trace of methylated coffee bud extract. Peak indicated Me ABA had a retention time and mass spectrum identical to authentic Me ABA

time as methyl abscisate (5.4 rain when run isothermally and 12.6 rain when the temperature was programmed). Co-injection of the methylated extract and ABA gave a single sharp peak. Identification was confirmed b y mass spectrometry. One of three peaks in the total ion current trace from a 8E33 column (see Fig. 1) had a mass spectrum identical with t h a t of authentic methylated ABA. The yield of ABA obtained from dormant flower buds was 0.1-0.16 ~tg/g dry weight as estimated b y bioassay compared to O . 2 i 0 . 3 ~ g / g dry weight estimated b y Optical R o t a t o r y Dispersion. Bioassay of the ABA obtained from bud material collected 2 days after bud break gave a yield of 0.04-0.09 Fg/g dry weight. ABA accounted for about 75 % of the inhibitory activity found in the acidic ethyl acetate extract. To decide whether endogenous ABA plays a part in flower bud dorm a n c y information is still required about its source, movement, specificity of response, and the relationship between concentration and effect (Jacobs, 1959). Furthermore, it is necessary to show that at bud break levels of endogenous ABA decrease or t h a t levels of endogenous bud-

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break-promoting substances increase. Even then, such evidence could not establish unequivocally t h a t a causal relationship exists between abscisic acid and dormancy in coffee flower buds. If ABA does play a part in flower bud dormancy, then the suggestion b y Alvim (1960) t h a t a water deficit is necessary to remove an inhibitor responsible for flower bud dormancy can be re-interpreted. The viability of Alvim's hypothesis is suggested b y certain other evidence. Thus, Alvim (1960) was able to increase the numbers of flowers opening in response to aqueous sprays of GA by increasing the soil moisture deficit. Again, in experiments (Browning, unpublished) where GA and BA were applied in microdrops of ethanol or as aqueous sprays to dormant flower buds, BA broke dormancy only after four weeks of drought, whereas GA was effective after a shorter period of drought. After one week of drought GA did not cause break but increased the numbers of flowers opening in response to rainfall. During drought the ability of dormant buds to flower in response to applied hormones clearly increases, and this could reflect declining levels of an inhibitor. I n contrast to the above hypothesis, Wright et al. (1969) found an increase in the absolute amount of ABA in wheat leaves which had been detached and then wilted, and they suggested t h a t water stress in the field m a y increase the ABA content of plant tissues. This evidence does not, however, preclude the possibility that the amount of ABA in dorm a n t coffee flower buds is lower after the tree has been exposed to drought. Changes in the ABA content of dormant buds collected at different times during drought are now being studied. Again, the more marked dormancy at high temperatures of coffee flower buds (Went, 1957) might also be mediated b y changing levels of abscisic acid. The authors wish to thank Dr. G. Ryback for the O. R. D. analysis; Dr. J. MacMillan and Dr. L. C. Luckwill for useful comments on the manuscript; and F. Hoffmann-La-Roehe& Co. for a sample of abscisic acid. G. B. was supported financially during this work by a Ministry of Overseas Development Studentship and P. G. by Tare & Lyle Ltd.

References Alvim, P. T. de: Estimulo de la floraciSn y fructificaciSn del Cafeto por aspersiones con s giber6lico. Turrialba 8, 64-72 (1958). - - Physiology of growth and flowering in coffee. Turrialba 2, 57-62 (1960). Bowen, M. R., Hoad, G. V. : Inhibitor content of phloem and xylem sap obtained from Willow (Salix viminalis L.) entering dormancy. Planta (Berl.) 81, 64-70 (1968). Cornforth, J. W., Milborrow, B. V., Ryback, G., Wareing, P. F. : Identity of Sycamore "Dormin" with abscisin II. Nature (Lond.) 205, 1269-70 (1965). Eagles, C. F., Wareing, P. F. : The role of growth substances in the regulation of bud dormancy. Physiol. Plantarum (Cph.) 17, 697-709 (1964).

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Jacobs, W. P. : What substance normally controls a given biological process? I. Formulation of some rules. Develop. Biol. 1, 527-533 (1959). Luckwill, L. C., Weaver, P., Macmillan, J. : Gibberellin and other growth hormones in apple seeds. J. Hort. Sci. 44, 421-24 (1969). Mes, M. G.: Studies on the flowering of Co//ea arabica (L.). New York: 1. B. E. C. Res. Inst. 1957. Milborrow, B. V. : The identification of (~-)-Abscisin I I [(+)-dormin] in plants and the measurement of its concentration. Planta (Berl.) 76, 93-113 (1967). Ohkuma, K., Lyon, J. L., Addicott, F. T., Smith, 0. E. : Abscisin I I - an abscission accelerating substance from young cotton fruits. Science (N. Y.) 142, 1592-1593 (1963). Pagacz, E. A. : Quelques considerations sur la floraison du caf6ier. Bull. agricole du Congo Belge 6, 1531-1538 (1959). Rees, A. R. : Some observations on the flowering behaviour of Co]lea rupestris in Southern Nigeria. J. Ecol. 5~, 1-7 (1964). Veen, R. van der: Plant hormones and flowering in coffee. Acta Bet. Neerl. 17, 373-76 (1968). Went, F. W. : The experimental control of plant growth. 343 pp. Waltham, Mass. : Chronica Botanica Co. 1957. Wormer, T. M. : Some physiological problems of coffee cultivation in Kenya. Caf4 6, 1 (1965). Wright, S. T. C., Hiron, R. W. P. : (~)-Abscisic acid, the growth inhibitor, induced in detached wheat leaves by a period of wilting. Nature (Lend.) 224, 719-720 (1969). Mr. G. Browning University of Bristol, tCesearch Station Long Ashton Bristol, BS 18 9AF, England