Planta (Berl.) 86, 63--68 (1969)

Studies on Abscisic Acid in Apple Seeds RYSZ~D RUDNICKI Research Institute of Pomology, Skierniewiee, Poland Received November 25, 1968/January 17, 1969 Summary. Abscisic acid (AbA) was found to be present in non-stratified apple seeds. The evidence is based on chromatographic behaviour, fluorescence, UVspectrum and growth inhibition in a wheat-coleoptile straight-growth test. The AbA level, as measured by this bioassay, falls considerably with progressing stratification ; after 3 weeks of after-ripening no AbA could be detected in the seeds. The longer the period of stratification, the higher was the concentration of synthetic AbA necessary to inhibit germination.

Introduction The seeds of apple require about 12 weeks of after-ripening at low temperature (stratification). During this time the seeds undergo certain, little-understood bioehemicM processes leading to the full mobilization of their food reserves necessary for normal growth and development of the seedlings. The regulation of these processes is undoubtedly controlled by hormones, and a promotor-inhibitor complex seems to play a key role (e.g. AMEN, 1968). Gibberellin and cytokinins have a stimulatory effect on the germination of app]e seeds (KAMINSKI and PIE~I4~EK, 1968) whereas some growth inhibitors such as chlorogenie acid (C6ME, 1967) and AbA inhibit their germination. PI]~NI4ZEK and RuD~ICKI (1967) found that an inhibitor similar to abseisic acid (AbA) was present in non-stratified whole apple seeds and that this compound strongly inhibited the germination of these seeds (see also KAMINS~:I, 1968). The physiological meaning of these findings was however not clear, and an attempt was therefore made to extend the information on the occurrence of AbA in apple seeds, and to investigate the quantitative changes in the AbA content of apple seeds during stratification as well as the sensitivity of apple embryos to the inhibitor after various lengths of cold treatment.

Material and Methods Seed Material. Seed of apple (Pyrus malu8 L., cv. Antonovka) collected in September 1967 at the Sinot@a Experimental Orchards of the Institute of Pomology, Skierniewice, were used for the extraction of AbA and in the germination tests.

64

R. I~UDNICKI:

Extraction and Identi/ieation o/ AbA /rom Apple Seeds. The seeds (500 g or ca. 14,000 seeds) were homogenized in 2 ltr of 80% ethanol. The insoluble material was removed by centrifugation and the alcohol evaporated under vacuum at 50 ~ The water fraction was adjusted to p H 3 with 10% HC1 and extracted for 24 hr with freshly distilled diethyl ether in a n apparatus for liquid-liquid continous extraction. The ether extract was t h e n shaken with a 4% sodium bicarbonate solution, and the emulsion left for 24 hr in a separatory funnel. The sodium bicarbonate fraction obtained was acidified to p H 3 and again extracted with diethyl ether. This ether extract was evaporated to dryness under nitrogen and the residue taken up in 80% ethanol. I t was then applied in a streak to W h a t m a n No. 3 paper. The chromatograms were developed by the ascending technique in isopropanol : amm o n i a : w a t e r 10:1:1 (v/v). Synthetic AbA was spotted at both ends of the streak as a marker. After development, the synthetic marker a n d natural AbA were detected under a "Mineralight" ultraviolet lamp and the natural AbA additionally measured b y the wheat bioassay according to the method of NITSC~ (1956), as described below. The strip of paper showing either UV-absorption, or m a x i m u m inhibition in the bioassay was eluted with 80% ethanol and the eluate evaporated under vacuum to a small volume. I t was further purified on thin-layer plates coated with Silica gel GF-254 (Merck), according to I~UD~ICK. and ANTOSZEWSKI (1968), by successive separation in the following Solvents (all v/v): Rf (AbA) 1. isopropano] : butanol : ammonia : water (6: 2 : 1 : 2) 0.65 2. benzene:ethyl acetate:acetic acid (70:30:5) 0.30 3. benzene:acetone:acetic acid (70:30: 1) 0.40 4. benzene:chloroform:formic acid (20:100:10) 0.12 These solvents resolved AbA from most of the impurities. The spots were eluted from the thin-layer plates with 1% acetic acid in methanol, and the eluates evaporated to a small volume under nitrogen at 30 ~ One aliquot of the highly purified extract was then assayed in a wheat eoleoptile test. Another aliquot was identified as AbA b y its blue-green fluorescence in U V when sprayed with sulphuric acid on thin-layer plates developed in solvent 2 in the presence of synthetic 1 AbA. This fluorescence was measured qaar~titatively by a spectrofluorimetric technique as described by ANTOSZEWSKI and I~UDNICKI (in preparation). I t was found t h a t the intensity of the blue-green fluorescence of a spot of the natural AbA from the extract could be compared with t h a t of spots of synthetic AbA at different concentrations. Thus, the fluorescence ,of a 0.1 ~g spot of synthetic AbA could be detected a n d compared with corresponding spots of natural AbA. Bioassay. The straight - - growth wheat - - coleoptile bioassay used was a modification of t h a t b y 1WITSC~ (1956), using a Polish variety (Tritieum acstivum L., cv. Ostka Chtopicka). 10 sub-apical sections 3.5 m m in length were incubated in 1 ml of 0.5% sucrose for 20 hr in darkness either with segments of paper chromategrams t h a t had been divided into .14, 2-cm strips, or with known concentrations of AbA (0.1--5 ~g ml-~). For the quantitative determination of AbA concentration in the highly purified extract from homogenized seeds, 12 geometric dilutions were used and the percentage of coleoptile-growth inhibition compared with a standard curve of activity for synthetic AbA. Inhibition of wheat-coleoptile growth caused b y 1 Izg m1-1 of AbA was still measurable. The lengths of sections were 1treasured under a stereomicroscope to 0.1 ram. The vertical scales of the histograms of Fig. 1 represent the section lengths as percent of controls; each bar is a mean of three chromatogram or extract coficentrations. 1. ( R S ) - A b A = ( ~ - ) - A b A ; natural A h A = ( S ) - - A b A = ( - - ) - - A b A .

Abscisic Acid in Apple Seeds

65

Changes o[ AbA Levels During Stratification. The changes in the level of AbA during the stratification of apple seeds were assessed on the basis of the wheatcoleoptilc test in samples composed of 800 seeds. Stratification was conducted in moist sand at 2 4 ~ and the samples were taken every week during a period of 10 weeks, beginning with non-stratified seeds. Whole seeds were extracted for 7 days at 4 ~ with 100 ml of distilled water changed daily, according to a method described by LIPE and CRA~ (1956). The combined water extracts were acidified to pH 3 with I0% tIC1, extracted directly with diethyl ether, and later processed in the same way as the water fraction from homogenized seeds. Paper ehromatography in isopropanol: ammonia:water (10:1 : 1, v/v) was used throughout, and no further attempt at purification of the inhibitor was made. Fig. 1 (A--I-I) shows the average results from 3 samples, each of 800 seeds and each extracted separately; values of the confidence interval at the 0.05 level of probability are indicated. Sensitivity o/ Apple Embryos to AbA. The sensitivity of apple embryos to synthetic AbA after various lengths of after-ripening at 2--4 ~ was investigated in the following manner. Non-stratified seeds and seeds after-ripened for 3, 6, 9 or 12 weeks were used. The seed coats (testa and endosperm) were removed and samples of 40 embryos were treated with synthetic AbA at concentrations from 0.03--4.0 t~g ml 1. Four ml of the appropriate test solution were poured into 9-em Petri dishes, 3 dishes being used per trea~men}. The control embryos were treated with the same volume of distilled water, The embryos were germinated in a growth chamber, at 24~ and in darkness, for 5 days. The number of germinated embryos with radicles longer than 3 mm were eoanted every 24 hr and the results are presented in the table as the mean sum of the daily percentage of germination for the 5-day period starting with the first day of imbibition, according to the method proposed by TIMso~ (1965). Thus 2J 5 =.,500 would mean 100% germination during the first day. Results A b o u t 20 ~zg of a highly purified inhibitor were o b t a i n e d from 500 g of non-stratified apple seeds. On the basis of its chromatographic behaviour on paper a n d on t h i n - l a y e r plates (R i values i n the presence of s y n t h e t i c A b A as a marker), the fluorescence test with sulphuric acid on the plates coated with Silica gel, the U V a b s o r p t i o n curve a n d biological a c t i v i t y in the wheat coleoptile straight growth test, the i n h i b i t o r was shown to be identical with AbA. Using a t least 10 geometrical dilutions with 3 replicates a t each concentration, the a c t i v i t y of the i n h i b i t o r was compared i n the w h e a t bioassay with k n o w n concentrations of synthetic AbA. I t was f o u n d t h a t i n 800 non-stratified seeds the i n h i b i t o r y a c t i v i t y of A b A (Fig. 1 A) was v e r y high. ~4dter 1 a n d 2 weeks of stratification the c o n t e n t h a d dropped b y ca. 20 % a n d 40 %, respectively, a n d after 3 weeks of stratification no A b A could be f o u n d i n the seeds (Fig. 1 D). The experiments on the response of apple embryos to exogenous A b A as related to after-ripening (Table) showed t h a t the lowest A b A c o n c e n t r a t i o n used (0.03 Bg ml-1) h a d very little effect on the germi5

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Fig. 1 A--I{. The changes in the level of abscisic acid in apple seeds, based on results with a wheat-coleoptile bioassay. A unstratified seeds; B after 1 week of stratification; C after 2 weeks; D after 3 weeks; E after 4 weeks; F after 6 weeks; G after 8 weeks; H after 10 weeks

Table. Germination o/ apple embryos measured as Z 5 alter di]/erent length o/

strati/ication Concentration of AbA (/~g m1-1)

Days of stratification 0

21

42

63

84

405 378 338 318 225 63 10 0 0

455 430 415 363 335 227 99 40 0

460 450 460 390 343 265 170 50 13

Germination as Z 5 0 (control) 0.031 0.062 0.125 0.25 0.5 1.0 2.0 4.0

0 0 0 0 0 0 0 0 0

123 94 80 28 3 0 0 0 0

Abscisic Acid in Apple Seeds

67

nation of the apple embryos, regardless of the period of stratification. Increasing AbA concentrations, however, had increasing inhibitory effect on embryo germination and, furthermore, the shorter the length of the stratification period, the greater was the sensitivity of the embryos to AbA. Thus, germination of embryos which had been stratified for 21 days was completely inhibited by 0.5 ~g ml -I AbA whereas germination of embryos stratified for 42 days did occur at this AbA concentration, but at a much reduced rate as compared to the untreated controls. Fully stratified embryos germinated slightly even at the highest AbA concentration used. Discussion

I t was found that AbA is present in apple seeds, and thus the preliminary identification of AbA in unhomogenized, non-stratified whole apple seeds (1)IE~I4ZEK and t~UD~ICKI, 1967) was confirmed. The results presented here showed that after 3 weeks of stratification AbA disappeared from the seeds (Fig. 1), and the sensitivity of apple embryos to AbA decreased in the course of after-ripening (Table). The actual concentration of AbA in the homogenized seeds was probably higher but in the first ether extract not all the inhibitor could be recovered from the emulsion, and further losses occurred during the repeated purification by paper and thin-layer chromatography. However, this strict purification left us with a sample of AbA that was free from other UV-absorbing substances. Disappearance of AbA from the seeds during stratification was reflected in their ability to germinate. Embryos stratified for 42 days germinated better than those stratified only 21 days. On the other hand, the growth of seedlings from embryos stratified for 21 and 42 days was still impaired.. The decrease in the sensitivity of embryos to exogenous AbA in the course of after-ripening suggests an increase in the activity of an endogenous mechanism metabolizing this inhibitor. The growth of the radicle was inhibited proportionally to the applied concentration of AbA (unpublished). At low concentrations of AbA, although the stratified seeds germinated normally, the subsequent early stages of growth were clearly inhibited (KA~I~sKr et al., 1968). I t seems that partly and fully stratified apple embryos are a very sensitive material for the study of the interaction of AbA with endogenous growth stimulators, and this is reported separately (Kh~I~SKI et al., 1968). However, the question remains as to why after 3 weeks of stratification, when most of AbA is absent from the seeds, and when they are able to germinate, their subsequent growth is inhibited. It is possible that only free AbA fluctuates during stratification and that 5*

68

}~. RUDNICKI:Abscisie Acid in Apple Seeds

other forms (KosHINIZU, 1968 ; GASKIN a n d MAcMmLA~, 1968 ; MACMILLAN a n d P~YCE, 1968) are present in the seeds. Nevertheless, the evidence reported i n this work shows t h a t A b A is the m a i n i n h i b i t o r of g e r m i n a t i o n i n apple seeds, a n d t h a t stratification leads to r e m o v a l of this inhibitor. This work was partly supported by the U.S. Department of Agriculture, Agricultural Research Service Grant No. FG-Po-199.

References AME~, R. D.: A model of seed dormancy. Bot. Rev. 34, 1--32 (1968). C(SME, D. : L'inhibition de germination des grMnes de pommier ,,Pirus malus L." non dormantes. R61e possible des ph6nols t6gumentaires. Ann. Sci. Nat. Bot. S6r. 8, 3 7 1 ~ 7 8 (1967). GASKI~, P., and J. MAcMILLAn: Identification and estimation of abscisic acid in a crude plant extract by combined gas chromatography-mass spectrometry. Phytochemistry. 7 1677--1701 (1968). KAMI~S~I, W. : Inhibitory effect of apple juice on the germination of apple and cherry seeds and the growth of apple seedlings. Acta Soc. Bot. Pol. 37, 173--178 (1968). --, and J. PIE~I4~K: The influence of growth regulators on the germination of the apple cultivar Antonovka seeds from which the seed coats were removed. Bull. Acad. Pol. Sci. 16, 719--723 (1968). - - R. RVD~ICKI, and J. P I E N I 4 ~ : The interaction of abscisic acid and growth stimulators in the germination of apple embryos and in seedlings growth. Internat. Syrup. of Seed Physiology of Woody Plants, K6rnik Arboretum (1968) (in press). Kos~IMIZU, K., M. INw, H. F i x v I , and T. MITS~I: Isolation of (+)-D-glucopyranoside from immature fruit of Lupinus luteus. Agr. Biol. Chem. 32, 789--791 (1968). LIe~, W. N., and J. C. C~A~E: Dormancy regulation in peach seeds. Science 153, 541--542 (1966). MACMILLAn, J., and R. J. PaYeE: Phaseic acid, a putative relative of abscisic acid, from seed of Phasedus multiflorus. Chem. Commun. 1968, 124--126. NITSC~, J. P. : Methods for investigation of natural auxins and growth inhibitors. In: The Chemistry and Mode of Action of Plant Crowth Substances (R. L. WAI~and F. Wm~TMA~, eds.), p. 3--39. London: Butterworths 1956. PIENI4~EK, J., and R. RuDNrCKI: The presence of abscisin II in apple leaves and apple fruit juice. Bull. Acad. Pol. Sci. 15, 251--254 (1967). RVD~ICKI, R., and R. ANTOSZEWSKI:The labelling of abscisie acid (dormin) with carbon-14 in strawberry by means of photosynthesis. Bull. Acad. Pol. Sei. 16, 447--449 (1968). TiMso~, I.: New method of recording germination data. Nature (Lond.) 207, 216--217 (1965). RYSZARD I~UDmCKI Department of Plant Physiology Institute of Pomology Skierniewice, Poland