Plants (BEE.) 78, 305--309 (1968)

Identification of (+)-Abscisic Acid in Strawberry Leaves 0LFAT M.K. GAB~ and C. G. GUTTttIDGE Scottish Horticultural Research Institute, Invergowrie, Dundee Received October 21, 1967

Summary. The acidic ether extract of leaves collected in October from dormant strawberry plants outdoors at Invergowrie inhibited coleoptilegrowth in germinating wheat embryos. On paper chromatograms developed either with isopropanoh ammonium hydroxide: water (10:1 : 1) or isopropanol: 1% ammonium hydroxide (4:1), the zones possessing inhibitory activity had Rf's 0.6--0.7 and 0.6--1 respectively. Fractions possessing comparable activity were eluted from granular animal charcoal columns by 10% and 20% acetone in water, and, on further purification, eluted only by 10% ethyl acetate in chloroform from columns of celitesilicic acid (2:1). The inhibitor in this fraction was identified as (+)-abseisic acid (abscisin II, dormin) by spectropolarimetry. Introduction Liv and CAI~S (1961) isolated an abscission-accelerating substance (abscisin) from cotton burs. Later another abscission-accelerating substance (abscisie acid or abscisin II) was isolated from young cotton fruits by 0I~KUMA et al. (1963) and its structure established (0HIiVMA et al., 1965). Independently RoBI~SOl~ and WAREI~G (1964) demonstrated the presence of a growth inhibiting compound in sycamore (Acer pseudoplatanu8) which induced dormancy. COl~I~rOl~Ttt etal. (1965b, 1966a) isolated and identified this compound as abseisie acid (abscisin II) and later COI~XFOI~TH et al. (1966b) used speetropolarimetry to identify abscisie acid in extracts of birch, sycamore, cabbage, potato, avocado (Persea gratissima) and lemon, and MiL]~om~ow (1967) has extended this list. Synthetic abscisin has half the inhibitory activity of the naturally occurring compound (Colz?CFOl~TI~et al., 1965a). After prolonged exposure to short photoperiods strawberry plants became dormant (DAR~OW, 1933). The dormant condition is charaeterised by restricted but not weak vegetative growth (GuTTRIDGE, 1958) and although there is no resting bud as in woody plants, there are obvious parallels, suggesting that abscisie acid or a similar compound might be present in leaves of dormant strawberry plants in autumn. Recently exogenous application of synthetic abseisic acid has been shown to induce flower formation in strawberry Fragaria ananassa Duch. (EL ANTABLu et al., 1967).

306

O . M . K . GABR and C. G. GUTTRIDGE:

Materials and Methods Leaves were collected in October 1966 from strawberry plants var. Talisman growing in the field at Invergowrie, frozen with solid carbon dioxide and stored at a temperature of - - 5 ~ C for various periods up to 7 months. 3 kg of leaves were thrice extracted with 80% methanol (RoBI~so:N and WA~EIXG, 1964). The extracts were t h e n pooled, the methanol evaporated under reduced pressure at a temperature not more t h a n 35 ~ C and the aqueous solution remaining stored overnight a t 3 ~ C. The aqueous extract was clarified b y centrifugation a t 5,000 r. p.m. for 10 minutes, the clear s u p e r n a t a n t solution was then adjusted to p H 3--3.5 with 5% sulphuric acid, extracted 4 times with ether, and the ether extracts combined a n d concentrated to a small volume. The concentrated ether extract was extracted 8 times alternately with saturated sodium bicarbonate and water; the ether solution t h a t remained is termed the neutral ether fraction. The pooled sodium bicarbonate a n d water fractions were acidified to pI-I 3--3.5 with 5% sulphuric acid a n d shaken successively with 4 lots of ether. The ether extracts were t h e n pooled and evaporated to dryness under reduced pressure a t a temperature not more t h a n 25 ~ C. This is termed the acidic ether fraction. The residue was dissolved in 10 ml acetone. The crude acidic ether extract was purified b y column chromatography using ]3. D.H. granular animal charcoal as an adsorbent and eluting with increasing proportions of acetone in water in 10% steps a n d finally with pure acetone. The fractions were bioassayed and those which showed inhibitory activity were combined a n d chromatographed on a column of celite (Hyflo supercel) a n d silicic acid (KochLight Laboratories) 2:1. The column was Muted with ethyl acetate in chloroform starting with 5%, then 10% and after t h a t in 10% steps ending with pure ethyl acetate. Each fraction was bioassayed and the active fractions combined and further purified on a second celite-silicic acid (2: 1) column and eluted with ethyl acetate in chloroform in 10% steps. P a p e r chromatography was done on W h a t m a n No. 1 paper with two solvent systems, isopropanol : a m m o n i u m hydroxide (sp. gr. 0.88) 1% solution 4:1 (EAGLES and WA~EINO, 1964) a n d isopropanol: a m m o n i u m hydroxide (sp.gr. 0.88): water 10:1:1 (HoAo, 1967). Each chromatogram was cut into l0 strips each equivalent to a n ]~f value of 0.1, and each strip was eluted with 2 ml water at 3 ~ C in the refrigerator. These aqueous extracts were bioassayed for inhibitory activity. ]3ioassays were done with wheat embryos (MIYAMOTO et al., 1961) and seeds of (Lepidium) Lepidium sativum (BAYE~, 1965). W h e a t ( Triticum vulgare var. Sveno) or .Lepidium seeds were sown in petri dishes (2 in. diameter) containing two 5.5 cm W h a t m a n No. 1 filter papers moistened with w~ter or with the solution to be assayed a n d the dishes incubated at 27 ~ C for 48 hours in darkness. W i t h wheat, the seeds were cut transversely and 13 halves containing the embryos were used for each test. After incubation the coleoptiles were measured to the nearest 0.5 m m and the results expressed as percentage length of coleoptiles of controls grown in water. W i t h Lepidium, 20 seeds were used for each assay a n d activity estimated from the percentage inhibition of germination compared with water controls.

Results and Discussion T a b l e 1 s h o w s t h e r e s u l t s of a s s a y i n g f r a c t i o n s f r o m p a p e r c h r o m a t o g r a m s of t h e a c i d i c - e t h e r e x t r a c t f r o m t h e e q u i v a l e n t of 30 g m . f r e s h w e i g h t of s t r a w b e r r y l e a v e s . W h e n t h e c h r o m a t o g r a m w a s d e v e l o p e d w i t h t h e ( 1 0 : 1 : 1 ) s o l v e n t a n d t h e a c t i v i t y of i n d i v i d u a l s e c t i o n s b i o a s -

Identification of (+)-Abscisic Acid in Strawberry Leaves

307

Table 1

Inhibitory activity o/ ]ractions o/ acidic ether extract o/ Talisman strawberry leaves

R~

Control 0.0--0.1 0.1--0.2 0.2--0.3 0.3--0.4 0.4--0.5 0.5--0.6 0.6--0.7 0.7-- 0.8 0.8~0.9 0.9--1.0

Isopropanol : ammonium hydroxide: water 10:1:1

Isopropanol: 1% ammonium hydroxide 4:1

mean a S.E.

mean a S.E.

6.5 8.1 7.8 9.2 6.4 7.9 4.6 3.5 5.6 9.3 10.6

0.9 0.7 0.8 0.7 0.6 0.7 0.3 0.2 0.3 0.8 0.8

% of P control

124~ 120 141 98 121 70 54 86 143 163

0.1 0.3 0.05 0.9 0.2 0.1 0.01 0.3 0.05 0.01

6.7 6.8 5.2 5.8 6.1 8.2 7.0 4.4 3.5 3.9 4.4

0.7 0.7 0.6 0.9 0.5 0.6 0.9 0.2 0.3 0.4 0.4

% of P control

101 77 86 91 122 104 65 52 58 65

0.9 0.2 0.5 0.5 0.3 0.7 0.01 0.01 0.01 0.02

The acidic-ether fraction was developed on Whatman No. 1 paper by the 10:1 : 1 or 4:1 solvent mixtures, and the individual sections of the chromatograms were bioassayed by the wheat test. a Mean length of 13 eoleoptiles.

sayed with the wheat test, i n h i b i t o r y a c t i v i t y was detected in a zone with R~ 0.6--0.7. W h e n e h r o m a t o g r a m s of the acidic ether fraction were developed with the (4 : 1) solvent i n h i b i t o r y a c t i v i t y was spread over a zone with Rf 0.6--1.0. No such a c t i v i t y was detected in the n e u t r a l ether fraction similarly analysed. The acidic ether extract was e h r o m a t o g r a p h e d on a c o l u m n of g r a n u l a r animM charcoal (KosHIMIZU et al., 1966) using different concentrations of acetone a n d water as eluates. Each eluatc was reduced to a small volume a n d the e q u i v a l e n t of 29 gm. fresh leaves was used for each bioassay. The results of the wheat test (Table 2) show t h a t the fractions eluted b y 10 a n d 20% acetone i n water were inhibitory. The fractions eluted b y 10, 20 a n d 30% acetone in water completely inhibited the g e r m i n a t i o n of Lepidium seeds. B y contrast, KOSHI3IIZU et al., 1966 f o u n d t h a t the i n h i b i t o r y a c t i v i t y from e x t r a c t of l u p i n was located in fractions eluted with water c o n t a i n i n g 55 to 60 % acetone. The active fractions from the charcoal c o l u m n were pooled a n d chrom a t o g r a p h e d on a column of celite-silicic acid 2:1 (KosmMIzu et al. 1966; ADDICOTT et al 1966) eluted with increasing proportions of ethyl acetate in chloroform. Each eluate was c o n c e n t r a t e d a n d the e q u i v a l e n t of 47 gm. fresh leaves used for each bioassay.

308

O . M . K . GA]~Eand C. G. GIITTI~IDGE:

Table 2. Wheat test o/ the di//erent ]raetions o/ the 3 columns used /or puri/ieation

o] the acidic ether extract Granular animal charcoal column

Celite-silicic acid first column

Celite-silicie acid second column

Eluate

Coleoptfle length

Eluate

Coleoptile length

Eluate

% acetone

% of control

% ethyl

% of control

in water

10 20 30 40 50 60 70 80 90 lO0

acetate in chloroform 32 64 88 100 103 94 109 101 108 88

5 10 20 30 40 50 60 70 80 90 100

73 65 61 82 101 78 100 102 98 95 106

Coleoptile length

% ethyl

% of

acetate in chloroform

control

10 20 30 40 50 60 70 80 90 100

36 97 91 89 74 91 78 87 107 89

Inhibitory activity was detected in fractions eluted with 5, I0, and 20% ethyl acetate in chloroform in the wheat test (Table 2), and the same fractions completely inhibited germination of Lepidium seeds. Under comparable conditions KOSHIMIZ[Y et al. (1966) found that the inhibitor was eluted in 25 % ethyl acetate in chloroform, while ADDICOTT et al. (1966) reported that the activity was found in the 10 to 30% ethyl acetate e]uates. Fractions showing inhibitory activity were combined, applied to a second celite-silicic acid column (2:1) and again eluted with increasing concentrations of ethyl acetate in chloroform in 10% steps. After concentrating, the elnates were bioassayed, the equivalent of 59 gin. fresh weight of strawberry leaves being used for each assay. This time activity was found only in the fraction eluted by 10% ethyl acetate in chloroform (Table 2). Using extracts from cotton ADDIeOTT et al. (1966) and OHKUM• et al. (1963) found that abscisin II was eluted by 10% ethyl acetate in chloroform from a silicie acid-eelite column. When synthetic abscisie acid was ehromatographed on paper with the two solvent systems used for the acidic ether extract of strawberry leaves and sections of the chromatograms bioassayed, using the wheat test, an inhibitory zone was detected at Rf 0.6--0.7 using the i0:I:I solvent and at 1%~ 0.6--0.9 using the 4 :1 solvent. As these results parallel those from the paper ehromatograms of the acidic ether fraction, they suggest that the extract of strawberry leaves in fact contained abseisic

Identification of (+)-Abscisic Acid in Strawberry Leaves

309

acid. This was confirmed by speetropolarimetric analysis of the purified extract of strawberry leaves eluted from the second celite-silicic acid column and a yield of about 1.3 ~zg of abscisic acid per gram dry weight of strawberry leaves was indicated. We wish to thank Dr. CORCeFORTI~for supplying synthetic abseisic acid and Dr. MILBORROWfor carrying out spectropolarimetrie analysis of the inhibitor.

References ADDICOTT,}O.W., 1~. OItXUMA, O.E. SMITH, and W.E. THIESSEN: Chemistry and physiology of abscisin II, an abscission-accelerating hormone. Natural pest control agents. Advanc. Chem. No 53, Amer. chem. Soc. 97--105 (1966). BAYER, 1VI.H. : Paper chromatography of auxins and inhibitors in two Nicotiana species and their hybrid. Amer. J. Bet. 52, 883--890 (1965). CORkXFORTH,J.W., B.V. MILBORROW,and G. RYBACK: Synthesis of (+)-abscisinII. Nature (Lend.) 206, 715 (1965a). , and P.F. WARm•G: Chemistry and physiology of "dormins" in sycamore. Nature (Lend.) ~05, 1269--1270 (1965b). Isolation of sycamore dormin and its identity with abseisin IL Tetrahedron, Suppl. 8, part II, 603--610 (1966a). - - - - Identification and estimation of (=k)-abscisin II (dormin) in plant extracts by spectropolarimetry. Nature (Lend.) 210, 627--628 (1966b). DA~l~OW,G.M., and G.F. WALI~O"Photoperiodism as a cause of the rest period in strawberries. Science, 77, 353--354 (1933). EAGLES, C.F., and P.F. WAI~m~G:The role of growth substances in the regulation of bud dormancy. Physiol. Plantarum (Copenh.) 17, 697--709 (1964). EL E~TA~LY,H.M.M., P.F. WAI~EINO, and J. HILL~AC;: Some physiological responses to DL abscisin. Planta (Berl.) 73, 74--90 (1967). OxUTTRIDG]~,C. G. : The effects of winter chilling on the subsequent growth and development of the cultivated strawberry plant. J. Heft. Sci. 33, 119--127 (1958). HOAD, G.V.: (+)-abscisin II ((+)-dormin) in phloem exudate of Willow. Life Sci. 6, 1113--1118 (1967). KosnlMIZU, K., H. FuKIN, T. MITSVI, and Y. OGAWr Identity of lupin inhibit~r with abscisin I I and its biological activity on growth of rice seedling. Agr. Biol. Chem. 30, 941--943 (1966). LIU, W.C., and H.R. CArats: Isolation of abscisin, an abscission accelerating substance. Science 134, 384--385 (1961). MILBORROW,B.V. : The identification of (+)-abscisin II ((+)-dormin) in plants and measurement of its concentration. Planta (Berl.) 76, 93--113 (1967). MIYA~OTO,T., Iq. E. TOLBm~T,and E.tt. Ev~I~soN: Germination inhibitors related to dormancy in wheat seeds. Plant Physiol. 36, 739--746 (1961). OtIKUMA,K., F.T. ADDICOTT,O.E. SMITH, and W.E. TtIIESSEN: The structure of abscisin II. Tetrahedron letters No 29, 2529~2535 (1965). - - J.L. LYow, F.T. A~DlCOT% and O.E. SMITE: Abscisin II, an abscission accelerating substance from young cotton fruit. Science 142, 1592--1593 (!963). RoBinson, R.M., and P.F. WAI~II~G: Chemical nature and biological properties of the inhibitor varying with photoperiod in Sycamore (Acer 2~seudoplatanus). Physiol. Plantarum (Copenh.) 17, 314--323 (1964). -

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Dr. C.G. GUTTRIDGE,Dr. O.M.K. GABI~ Scottish HorticulturM Research Institute Invergowrie Dundee, Scotland 21a Planta(Berl.),Bd. 78

Identification of (+)-abscisic acid in strawberry leaves.

The acidic ether extract of leaves collected in October from dormant strawberry plants outdoors at Invergowrie inhibited coleoptile growth in germinat...
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