Planta (Berl.) 111,347--352 (1973) 9 by Springer-Verlag 1973

Identification of Gibberellin A20, Abscisic Acid, and Phaseic Acid from Flowering BryophyUumdaigremontianum by Combined Gas Chromatography-Mass Spectrometry P. Gaskin and J. MacMillan School of Chemistry, University of Bristol, Bristol BS8 1TS., U,K. J. A. D. Zeevaart MSU/AEC Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48823, USA Received March 9, 1973

Summary. The presence of abscisic and phaseic acid in a purified acidic extract from flowering plants of the long-short-day plant Bryophyllum daigremontianum [(R. Itamet and Perr.) Berg.] was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of their methyl esters. Gibberellin A~o (GA2o) was identified by GC-MS of the methyl ester and the trimethylsilyl ether of the methyl ester. The following levels of the 3 compounds per kg fresh weight were estimated: Abscisic acid, 5.5 ~g; phaseic acid, 9.4 txg; gibberellin A20, 0.8 t~g. When GA20 and four other GAs were applied to Bryophyllum under shortday conditions, the order of effectiveness for induction of flower formation was: GAs> GAI> GA5-= GAv> GA20. The low biological activity of the native GA20 is discussed. Introduction Acidic extracts of the long-short-day plant Bryophyllum daigremontianum [(R. H a m e t and Perr.) Berg.] contain two gibberellins (GAs), one with GAz- , the other with GAs-like properties (Zeevaart, 1969b, c). The level of the GAs-like material increased considerably when flower formation was induced b y the shift from long-day (LD) to shor~-day (SD) conditions, and reached a m a x i m u m at the time flower primordia became visible ca. 35 days after the start of SD treatment (Zeevaart, 1969c). Since a high level of GA in the leaves of this species is a prerequisite for production of the floral stimulus (Zeevaart, 1969c), it was of interest to identify the endogenous GAs. We report here the identification of the GAs-like material as GA20, as well as the presence of abscisic acid (ABA) and phaseic acid in a partially purified extract

348

P. Gaskin et al. :

p r e p a r e d f r o m flowering Bryophyllum p l a n t s . F u r t h e r m o r e , t h e biological a c t i v i t y of t h e n a t i v e GA~0 a p p l i e d t o Bryophyllum u n d e r S D is c o m p a r e d w i t h t h a t of several o t h e r GAs.

Materials and Methods Plant Material Plants of Bryophyllum daigremontianum [(R. Hamet and Pert.) Berg.] were grown in a greenhouse under a 20-hr photoperiod as described previously (Zeevaart, 1969a). In order to induce flower formation, plants which had developed at least 20 leaf pairs, were transferred from the greenhouse to SD conditions in a growth room with 8 h light from fluorescent and incandescent lamps (intensity 3000 fc at 23 ~ and 16 h of darkness at 15~ Various groups of plants were harvested towards the end of the light period after exposure to 30-44 SD when flower primordia were macroscopically visible. The shoot tips and 3-4 uppermost leaf pairs with their axillary shoots were harvested, frozen in liquid N~, and lyophilized. A total of 922 g dry material (ca. 16 kg fresh weight) were collected from 197 plants.

Application o / G A s GAs were injected into the 4th leaf pair counted from the tip with the aid of a 10O-~l Hamilton syringe as described previously (Zeevaart, 1969a). Leaf pairs 2 and 3 were removed. Floral stages (0 ~ vegetative, 6 ~ normal inflorescence) were assigned as before (Zeevaart, 1969b).

Extraction and Puri/ication Procedures The dry material was extracted in 50-g batches and the acidic fraction from each batch was prepared separately following the procedures described for spinach (Zeevaart, 1971). Further purification of the acidic material took place in 4 successive steps: 1. Charcoal Chromatography. The ethyl acetate containing the acidic fraction was reduced in vacuo to a small volume, 20 ml of distilled water was added, and the rest of the organic solvent evaporated. The aqueous residue was pipetted on top of a charcoal-celite 535 (1:2, w/w) column and eluted with 80% acetone (Zeevaart, 1971). 2. Siliciv-acid Adsorption Chromatography. The acidic fraction obtained in step 1 was next run via a silicic acid-celite 535 (1:2, w/w) column using 2.5 g of silicie acid (Mallinckrodt, St. Louis, Mo., U.S.A., 100 mesh) for 50 g of dry material extracted. The acidic fraction was eluted with ethyl acetate-chloroform (1 : i, v/v). 3. P V P Chromatography. Insoluble polyvinylpyrrolidone (PV-P) obtained from the GAF Corp., New York, N.Y., U.S.A., under the brand name Polyclar AT, was screened (100-mesh sieve) and washed with 0.1 M phosphate barfer at pH 8.0, The washed PVP was slurried in the same buffer to set up a 30 • 1.9 cm column (Glenn et at., 1972). All acidic material obtained in step 2 was pooled, taken to dryness, and redissolved in 5 ml 0.5 M phosphate buffer at pH 8.0. The acidic fraction dissolved in buffer was pipetted on top of the PVP column and eluted with 0.1 M phosphate buffer (pH 8.0). The first 50 ml of effluent were discarded; the next 200 ml were collected (Glenn et al., 1972). After lowering the pH of the buffer to 2.5 with 6 N HC1, the acidic fraction was prepared by partitioning 5 times with half the volume of ethyl acetate.

GibberellinA~0in Bryophyllum

349

4. Siliciv-acid Partition Chromatography. The acidic fraction obtained in step 3 was further fractionated on a 5-g silieic-acid partition column (Powell and Tautvydas, 1967). The column was eluted stepwise with 15, 30, 40, and 50% ethyl acetate in n-hexane. These steps were chosen because it was established in a preliminary experiment that the main GA-like activity was present in the fractions eluted with 25 and 30 % ethyl acetate in n-hexane. 5. Thin-Layer Chromatography (TLC). Fraction 2 was crystallised from aeetone-hexane to give crystalline material (11.7 nag) m.p. 265-272 ~ which was a mixture by gas chromatography (GC) composed of non-gibberellins by mass spectrometry (MS). Half of the gum (12 mg) recovered from the crystallisation mother liquors was subjected to TLC on plates (20 • 20 cm) of silica gel ttFu54+a65 which had been pre-washed with ethyl acetate. The plate was developed with ethyl acetate-chloroform-acetic acid (15:5:1) and UV quenching zones were visualised by brie] exposure to UV light at 254 nm. Bands 1 to 5 at Rf values 0.30-0.37, 0.39-0.45 (non-quenching), 0.45-0.51, 0.51-0.57, and 0.57-0.62 were collected, and eluted with ethyl acetate. Band 1 (3.7 nag) crystallised on standing, showed two peaks after methylation by GC on 2% QF-1 at 210~ and was identical to the crystalline material described above. Bands 2, 3, and 4, which had the same Rf values as phaseic acid, GA5/20, and abscisic acid, respectively, were methylated and, in the case of band 3, methylated and trimethylsilylated, for examination by combined gas chromatography-mass spectrometry (GC-MS). 6. Combined Gas Chromatography-Mass Spectrometry (GC-MS). A GEC-AEI MS 30 mass spectrometer was used coupled to a Pye-Unicam 104 gas chromatograph through a silicone-membrane molecular separator. The mass spectra were determined at 24 eV with a source temperature of 210~ and a separator temperature of 185~ and they were recorded at 3 see per decade. For GC a glass column (5' • 1/3" of 2 % SE-33 on Gasehrom Q (80-100 mesh) was used with an He flowrate of 30 ml/min. The methylated bands 2 and 4 were chromatographed isothermally at 180~ the methylated and methylated-trimethylsilylated samples from band 3 were run at 210~ Bioassay Small aliquots of the fractions eluted from the silicic-acid partition column (step 4) were assayed on the d-5 mutant of corn (Zea mays L.) and the GA content expressed as f~g of GA3-equivalents (Zeevaart, 1971). Results and Discussion

Identi/ica~ion o/ A B A , Phaseic Acid and GA20 F r a c t i o n 2, einted with 30% e t h y l acetate i n n - h e x a n e from t h e silieic acid p a r t i t i o n c o l u m n (step 4), weighed 37.5 m g a n d c o n t a i n e d a t o t a l of 9 fzg GAa-equivalents as e s t i m a t e d i n t h e d-5 corn bioassay. Less t h a n 0.1 ~g GAa-equivalents were present i n the other fractions. Following TLC of fraction 2 (step 5) GC-MS of t h e m a j o r peaks i n t h e m e t h y l a t e d b a n d s 2 a n d 4 gave mass spectra which were identical with those of t h e a u t h e n t i c m e t h y l esters of phaseic (MacMillan a n d Pryce, 1969) a n d abscisie (Most et al., 1970) acids, respectively. The t o t a l i o n - c u r r e n t traces o b t a i n e d for t h e m e t h y l a t e d a n d for t h e m e t h y l a t e d - t r i m e t h y l s i l y l a t e d b a n d 3 are shown i n Fig. 1. I n each

P. Gaskin et al.:

350

T

0

Time,

fill

Fig. 1. Logarithmic total ion current (TIC) trace for band 3 (a) methylated, and (b) methylated and trimethyl silylated. 2 % SE-33 column at 210~

case the mass spectra obtained by scanning the arrowed peaks were identical to the spectra of authentic specimens of GA20 (Binks et al., 1969). B y quantitative GC using authentic phaseic, abseisie acids, and GA5 as calibration standards, the following total quantities were estimated: phaseic acid 150 ~g (9.4 ~g per kg fresh weight); ABA, 88 ~g (5.5 ~g per kg) ; and GA20 12 ~g (0.8 ~g per kg). The amount of GA~0 estimated b y GC (12 ~g) and bioassay (9 ~g) agreed closely, as can be expected since GA 3 and GA2o are about equally active in the d-5 bioassay (Crozier et al., 1970).

E//ects o] Applied GAs I t was established earlier (Zeevaart, 1969b) that 5 ~g GAa was the minimal dosage necessary to induce normal flowering in Bryophyllum daigremontianunt under SD. Using this treatment as a reference, different dosages of 4 other GAs, including GA~0, were applied to plants in SD. Since the experiments were conducted under controlled conditions (Zeevaart, 1969a)where the effects of GAa on stem growth and flowering are highly reproducible, only the pertinent data of 3 separate experiments arc presented (Table 1). The following were the minimal dosages of each GA required per plant to induce formation of normal inflorescences: 5 ~g GA S, 30 ~g GA1, 100 ~g GA~, 100 ~g GAv and ~ 100 ~g GA20. Thus, the order of effectiveness of the five GAs tested was: GAa > GA1 > GA 5 ~ GA~ > GA~o. Michniewicz and Lang (1962) working with Bryophyllum crenatum found that GAa and GA~ were equally active in causing flower formation and stem growth while GA1 and GA5 were 10 times less active.

Gibberellin A20in Bryophyllum

351

Table 1. The effect of various dosages of five different gibberellins on flower formation and s~em growth in Bryophyllum dalgremontlanum grown under shortday conditions GAs injected into 4th leaf pair from the tip. Leaf pairs 2 and 3 removed. GA applied per plant (t~g)

Flowering quotient a

Floral stage

Days until appearance of flower buds

Stem growth after 70 days (cm)

Control

0/5

0.0

co

13.8

GA1 10 30 100

2/5 5/5 5/5

1.6 6.0 6.0

56 39 36

21.5 46.8 70.8

GAs

2 5 20

0/5 5/5 5/5

0.4 6.0 6.0

co 39 36

19.9 37.6 68.0

GA5 30 60 100 GA~ 20 60 100

0/5 2/5 5/5 0/5 3/4

5/5

0.0 2.4 5.0 0.0 3.5 5.4

0o 49 38 co 39 36

21.7 28.2 51.2 17.4 28.0 36.4

GA~030 60 100

0/5 1/4 3/5

0.0 2.3 4.4

oo 63 51

21.6 25.0 34.1

a :Number of plants with flower buds/number of plants observed.

I t is surprising that the native GA~0 was least active in causing flower formation in Bryophyllum daigremontianum (Table 1), since the level of this GA is correlated with growth and developmental responses in this species (Zeevaart, 1969c). This shows that the relationship between the plant's growth response and endogenous hormones is not always straightforward. One possible explanation is that GA20 itself has little or no biological activity in BryophyUum, but must be converted to a GAs-like material (Zeevaart, 1969c) to become active. Unfortunately, the GAs-like materiM is always present in much smaller quantities than GA~0 so that its identification is not feasible because of the large amounts of plant material needed for extraction. There is, however, another possible approach to this problem: GA~0 can be synthesized by the eatMytie reduction of GA5 (Murofushi et al., 1968). By using tritium instead of hydrogen it is possible to prepare aH-GA20 by this method. Thus, the metabolism of radioactive GA20 can be studied in Bryophyllum, including the possible conversion to a biologically more active form.

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P. Gaskin et al. : Gibberellin A20in Bryophyll.ttm

Dr. N. Takahashi, Department of Agricultural Chemistry, The University of Tokyo, Tokyo, kindly donated a sample of GA20. The technical assistance of T. Kivilaan with extractions and bioassays is gratefully acknowledged. J. MacM. thanks the Science Research Council for a grant towards the purchase of the GEC-AEI MS 30 and for a technical assistant. J.A.D.Z. was supported by the U.S. Atomic Energy Commission under Contract AT(11-1)-1338.

References Binks, R., MacMillan, J., Pryce, R . J . : Plant hormones. VIII. Combined gas chromatography-mass spectrometry of the methyl esters of gibberellins A1 to A24 and their trimethylsilyl ethers. Phytochem. 8, 271-284 (1969). Crozier, A., Kuo, C.C., Durley, 1~. C., Pharis, R. P. : The biological activities of 26 gibberellins in nine plant bioassays. Canad. J. Bot. 48, 867-877 (1970). Glenn, J. L., Kuo, C. C., Durley, I~. C., Pharis, 1~. P.: Use of insoluble polyvinylpyrrolidone for purification of plant extracts and chromatography of plant hormones. Phytochem. 11, 345-351 (1972). MacMillan, J., Pryce, R. J. : Plant hormones. X. The constitution of phaseic acid; a relative of abscisic acid from Phaseolus multiflorus. An interpretation of the mass spectrum of phaseic acid and a probable structure. Tetrahedron 25, 5903-5914 (1969). Michniewicz, M., Lang, A. : Effect of nine different gibberellins on stem elongation and flower formation in cold-requiring and photoperiodic plants grown under non-inductive conditions. 1)lanta (Berl.) 58, 549-563 (1962). Most, B. H., Gaskin, P., MacMillan, J. : The occurrence of abscisie acid in inhibitots B1 and C from immature fruit of Ceratonia siliqua L. (Carob) and in commercial carob syrup. Planta (Berl.) 92, 41-49 (1970). Murofushi, N., Takahashi, N., u T., Tamura, S. : Gibberellins in immature seeds of Pharbitis nil. Part I. Isolation and structure of a novel gibberellin, gibberellin A20. Agr. Biol. Chem. 32, 1239-1245 (1968). Powell, L.E., Tautvydas, K . J . : Chromatography of gibberellins on silica gel partition columns. Nature (Lond.) 213, 292-293 (1967). Zeevaart, J. A. D. : The leaf as the site of gibberellin action in flower formation in Bryophyllum daigremontianum. 1)lanta (Berl.) 84, 339-3~7 (1969a). Zeevaart, J. A. D.: Gibberellin-like substances in Bryophyllum daigremontianum and the distribution and persistence of applied gibberellin Aa. Planta (Berl.) 86, 124-133 (1969b). Zeevaart, J. A. D. : Changes in the gibberellin content of Bryophyllum daigremontianum in connection with floral induction. Neth. J. agric. Sci. 17, 215-220 (1969c). Zeevaart, g. A. D. : Effects of photoperiod on growth rate and endogenous gibberellins in the long-day rosette plant spinach. Plant Physiol. 47, 821-827 (1971).

Identification of gibberellin A20, abscisic acid, and phaseic acid from flowering Bryophyllum daigremontianum by combined gas chromatography-mass spectrometry.

The presence of abscisic and phaseic acid in a purified acidic extract from flowering plants of the long-short-day plant Bryophyllum daigremontianum [...
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