Properties of Gibberellins from Flowering Plants CHARLES A. WEST a n d T H O M A S REILLY Department of Chemistry, University of California, Los Angeles 24, Calif.
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A gibberellin-like substance has been partially purified from acetone-water extracts of Lupinus succulentus seed and pods.
Its chromatographic
properties suggest a relatively polar gibberellin, perhaps identical with or closely related to the recently discovered gibberellin
A.
Purification
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of the gibberellin-like substances of Echinocystis macrocarpa endosperm led to the fractionation of two biologically active substances. Although there are insufficient data on which to base an identification, the chromatographic and fluorescence properties of Echinocystis Factor I most closely resemble those of the recently reported gibberellin A . The 7
chromatographic and fluorescence properties of Echinocystis Factor II resemble most closely those of gibberellic acid, but small differences suggest that these substances are not identical.
several years ago our laboratory investigated the presence of substances with * gibberellin-like biological activity in extracts of flowering plants, using the growth response of dwarf mutants of maize as a bioassay (9). Approximately one third of the crude extracts prepared from seed or fruit of Angiosperms gave evidence of such substances. A number of the active extracts were then chromatographed on paper and the active zones located by bioassay. The results suggested the presence of a family of gibberellin-like substances, since several of the R values of active substances differed significantly from one another and from those of the then known fungal gibberellins—gibberellins A and A and gibberellic acid. Subsequently, gibberellin A (I) and gibberellin A (II), a compound which has not yet been found in fungal filtrates, were isolated and identified as natural constituents of bean seed [from Phaseolus vulgaris (11) and Phaseolus muliflorus (6, 7)]. MacMillan, Seaton, and Suter (5) have recently reported the isolation and identification of two additional new gibberellins, A and A (III), from extracts of Phaseolus multiflorus seed. In this paper we describe the progress made in the identification of the gibberellin-like substances in extracts of two other plant species. f
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Gibberellin-like Substance of Lupinus succulentus Dough The gibberellin-like substance in acetone-water (1:1) extracts of the young seed and pods of Lupinus succulentus Dougl. did not migrate as far as gibberellic acid in any of a number of solvent systems used for paper chromatography. The ratio of the distance of migration of this gibberellin-like substance to that of gibber ellic acid in the same solvent system, the R value, was 0.44 in η-butyl alcohol1.5N ammonium hydroxide (3:1) (upper phase), 0.83 in pyridine-n-amyl alcoholwater (35:35:30) (upper phase), and 0.79 in η-butyl alcohol-glacial acetic acidwater (19:1:6) (upper phase). This behavior was unique among the gibberellins and plant extracts tested and suggested the presenpe of a more polar gibberellin in lupine extracts. Recently gibberellin A (III) has been isolated from bean extracts (5) and shown to have three hydroxyl groups. Since the other known gibberellins have only two or less hydroxyl groups and the same functional groups otherwise, A is the most polar of the gibberellins. Although there is no more direct evidence than R values at present, it may develop that the lupine gibber ellin-like substance is identical or closely related to gibberellin A . To gain further evidence on this point a large quantity of lupine seed and pods has ben extracted and the gibberellin-like substance extensively purified by solvent partitioning and charcoal adsorption and chromatography. However, the best fractions available are still impure, so that little more of its properties has been learned to date. ga
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Gibberellin-like Substances of Echinocystis macrocarpa Greene Both the endosperm and cotyledons of the wild cucumber, Echinocystis macrocarpa Greene, have been shown to contain gibberellin-like substances. The 38
WEST AND REILLY
Gibberellins
from Flowering
Plants
R value was determined as 1.9 in η-butyl alcohol-1.5N ammonium hydroxide (3:1) (upper phase) and 1.3 in pyridine-n-amyl alcohol-water (35:35:30) (upper phase). Somewhat later it was shown that gibberellin A (IV), a metabolite some times found in culture filtrates of Gibberella fujikuroi (10), gives these same R values in these solvent systems. Furthermore, Bukovac and Wittwer (I) and Lockhart and Deal (3) demonstrated that a number of species of Cucurbitaceae were much more sensitive in their response to A than to gibberellic acid or the other fungal gibberellins. These findings prompted the investigation of the chemical nature of the gibberellin-like substance of E. macrocarpa, a member of the family Cucurbitaceae. ga
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Purification The following scheme was followed in purifying gibberellin-like substances from E. macrocarpa. Approximately 2 liters of viscous endosperm extracted from immature seed was adjusted to p H 3 by the addition of sulfuric acid. This suspension was extracted directly several times with ethyl acetate. Most of the biologically active material was removed to the ethyl acetate phase, as detennined by bioassay with dwarf mutants of maize (8). The combined extracts were concentrated and extracted in turn with 5% aqueous sodium carbonate solution to remove acidic substances. All the biologically active material was removed to the aqueous phase. After this fraction had been acidified to p H 3, it was again extracted with ethyl acetate, which removed the biologically active substances to the ethyl acetate phase. The residue from the ethyl acetate layer (2.5 grams) was then chromatographed on a charcoal-Celite (1:2) column developed with increasing concentra tions of acetone in water. Fractions brought off the column with elutants ranging from 80% acetone in water to pure acetone (108 mg. of solids) contained most of the biologically active material. The solids in these fractions showed a yellow fluorescence when dissolved in concentrated sulfuric acid. Gibberellic acid (V) shows a similar fluorescence under these conditions, whereas gibberellin A and other fungal gibberellins do not. One-hundred-microgram portions of the biologically active solids from the charcoal column were chromatographed on paper using η-butyl alcohol-1.5N ammonium hydroxide (3:1) (upper phase) as the developing solvent. One chro matogram was tested for the presence of fluorescent materials after treatment with concentrated sulfuric acid and a second was tested for the presence of biologically active materials, using the dwarf maize mutant assay. Two zones of biological activity were located, both of which were correlated with wealdy fluorescent zones on the chromatogram. The slower-moving component behaved like gibberellic acid in its fluorescence and was only slightly displaced from it in position. The faster-moving component was very similar in position to gibberellin A ; however, A does not fluoresce under these conditions. The presence of a gibberellic acid-like material in this fraction was surprising, as there had been no indications of such a substance in crude extracts. It seemed possible that it had arisen from the faster-moving component as an artifact of chromatography. However, when the faster-moving component was eluted from a chromatogram developed with the η-butyl alcohol-ammonium hydroxide solvent system and rechromatographed in the same solvent system, there was no evidence of the formation of the slower-moving component from the faster one. The active solids from the charcoal column were subjected to a fractional 4
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precipitation by progressively increasing the concentration of petroleum ether (Skellysolve B) in a solution of the active material in ethyl acetate-acetone. Seventy-niné milligrams of white, amorphous material which showed some biological activity and fluorescence in sulfuric acid was precipitated first. A n apparently crystalline fraction which had biological activity and fluoresced in sulfuric acid solution was obtained next. This fraction yielded 20 mg. of solid on recrystallization from ethyl acetate-petroleum ether mixtures. A quantitative bioassay on maize mutants showed that this material was only about 2% as active as gibberellic acid on a weight basis. A paper chromatographic study revealed that both active zones were still present. Therefore, this fraction was subjected to further purification. Eight milligrams of the crystalline fraction was chromatographed on a column of anhydrous silicic acid developed with increasing concentrations of ethyl acetate in chloroform by a gradient elution technique. Fractions 6 and 7 contained 2.8 mg. of a biologically active white solid which showed a weak fluorescence in sulfuric acid. This material is referred to in subsequent discussion as Echinocystis Factor I. Fraction 13, eluted with a higher concentration of ethyl acetate in chloroform, contained 2.9 mg. of a biologically active, white solid which also showed a weak fluorescence in sulfuric acid. This material is referred to subsequently as Echinocystis Factor II. Properties of Echinocystis Factors I and II It is not known at present whether these preparations of Echinocystis Factors I and Π represent pure substances. The relatively weak fluorescence and biological activity of each preparation make it seem likely that they are still impure. Echinocystis Factor I behaves like gibberellin A on paper chromatography. No accurate quantitative measurements of its biological activity on the various dwarf mutants of maize have been made as yet, but it appears to be of a low order of magnitude in this assay as compared with gibberellic acid on a weight basis. The activation and emission spectra of Echinocystis Factor I in concentrated sulfuric acid have been measured in an Aminco-Bowman Spectrophotofluorimeter. The characteristic maxima are recorded here, with those of gibberellic acid for comparison. 4
Echinocystis Factor I Echinocystis Factor II Gibberellic acid
Fluorescence Maxima τημ Activation Emission 305,465 510-515 305, 330, 435 510-515 305,435 510
The second activation maximum for Echinocystis Factor I is approximately 30 τημ higher than that of gibberellic acid. The intensity of fluorescence is only about 3% that of gibberellic acid. Microspectrophotometric techniques for the determination of the acid equivalent weight (11) and of ethylenic double bonds (11) give a ratio of 0.75 double bond per acid equivalent in this fraction. Echinocystis Factor II behaves like gibberellic acid on paper chromatography. It also seems to have a low biological activity on dwarf mutants of maize when compared with gibberellic acid on a weight basis. It gives a weak fluorescence in sulfuric acid solution which is qualitatively very similar to that of gibberellic acid. The only detectable difference is the presence of an extra-weak activation maximum in the Echinocystis Factor II spectrum at 330 nv*. The intensity of the 40
WEST AND REILLY
Gibberellins
from flowering
Plants
flourescence of the Echinocystis Factor II sample is only about 3% that of gibber ellic acid. Microspectrophotometric tests suggest the presence of 0.90 ethylenic double bond per acid equivalent in this sample. Discussion The data currently available are an insufficient basis for definite conclusions regarding the structures of the gibberellins from Echinocystis. Echinocystis Factor I most closely resembles in its chromatographic and fluorescence properties gibberellin A (VI), recently reported as a fungal metabolite by MacMillan et al. (2). That it might be identical with A is an interesting possibility, since cucurbits are reported (2, 5) to be even more sensitive in their response to gibberellin A than to gibberellin A , which in turn is much more effective than the other fungal gibberellins (I, 3). If, in fact, these are the same, the sample of Echinocystis Factor I available for study must still be impure, since the fluores cence intensities of gibberellin A and gibberellic acid are of the same order of magnitude (4). Final identification of Echinocystis Factor I must await the preparation of a sample of known purity and a study of its properties. Echinocystis Factor II closely resembles gibberellic acid in its chromatographic and fluorescence properties, but the small differences noted suggest that they are not identical. The fact that there is no chromatographic evidence of Echinocystis Factor II in the crude extract used as starting material suggests that it may have arisen from Echinocystis I during the purification procedure, although there is no direct evidence to support this hypothesis. 7
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Acknowledgment The authors thank Fung L i u for valuable technical assistance throughout this work.
Literature Cited (1) Bukovac, M. J., Wittwer, S. H., ''Comparative Biological Activities of the Gibberel lins" in "Plant Growth Regulation" (Proc. of IVth International Conf. on Plant Growth Regulation), Iowa State University Press, Ames, Iowa, in press. (2) Cross, Β. E., Grove, J. F., McCloskey, P., MacMillan, J. Moffatt, J. S., Mulholland, T. P.C.,ADVANCESINCHEM.SER., No. 28, 3 (1961). (3) Lockhart, J. Α., Deal, P. H., Naturwissenschaften, 47, 141 (1960). (4) MacMillan, J., Akers Research Laboratories, Imperial Chemical Industries, Ltd., The Frythe, Welwyn, Herts, England, private communication. (5) MacMillan, J., Seaton, J. C., Suter, P. J.,ADVANCESINCHEM.SER.,No. 28, 18 (1961). (6) MacMillan, J., Seaton, J. C., Suter, P. J., Proc. Chem. Soc. (London) 1959, 325. (7) MacMillan, J., Suter, P. J., Naturwissenschaften 45, 46 (1958). (8) Neely, P. M., doctoral thesis, University of California, Los Angeles, 1959. (9) Phinney, B. O., West, C. Α., Ritzel, M., Neeley, P. M., Proc. Natl. Acad. Sci. U. S. 43, 398 (1957). (10) Takahashi, N., Seta, Y., Kitamura, H., Sumiki, Y., Bull. Agr. Chem. Soc. Japan 21, 396 (1957). (11) West, C., "Chemistry of Gibberellins from Flowering Plants" in "Plant Growth Regulation" (Proc. of IVth International Conf. on Plant Growth Regulation), Iowa State University Press, Ames, Iowa, in press. RECEIVED October 14, 1960. Research supported in part by National Science Foundation Grant 3526 and U. S. Public Health Service Grant 7065.
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