PACIFIC SOUTHWEST ASSOCIATION O F CHEMISTRY TEACHERS GIBBERELLINS AND PLANT GROWTH' CHARLES WEST University of California, Los Angeles
RECENTLY a group of substances known as gibberellins has attracted special attention because of the striking effects they have on growth and development when applied in small quantities to many types of flowering plants. I n fact, it is hoped by many that the external application of gibberellins may have large-scale uses in the management of economic crops. These gibberellins are compounds isolated from the culture filtrate of a particular fungus. There now is evidence, as will be discussed below, that substances having similar biological properties also occur naturally in flowering plants. Therefore, studies with gibberellins may also provide information concerning the natural regulation of growth and development of flowering plants. The general surge of interest in the gibberellins has come only recently even though their biological effects have been recorded for some time. A crystalline material was isolated from the culture filtrate of a fungus, Gibberella fujikuroi, by Yabuta and Sumiki a t the University of Tokyo in 1938 ( I ) . This fungus had earlier been shown to be the cause of a common disease of rice plants in the Orient characterized by overgrowth of the seedling stem and leaf parts followed by reduced yields of seed or, in severe cases, death before flowering. Yabuta and his co-workers demonstrated that microgram quantities of their crystalline material, which they named gibberellin A, could produce similar overgrowth of rice plants as well as of a number of other plant species tested. The Japanese group has continued work on the chemical and biological properties of gibberellin A to the present, but, in spite of the interesting implications of their experiments, similar studies mere not undertaken in laboratories outside of Japan until the past ten years. Then Mitchell and Angel a t Camp Detrick (8) and subsequently St,odola and co-workers of the United States Department of Agriculture (5) and, independently, Borrow,et al., a t Imperial Chemical Industries in Great Britain (4) undertook the production and isolation of gibberellin and a study of its properties. Currently research in this area is proceeding a t an accelerating pace in government, university, and private laboratories t,hroughout the world. A general review of the gibberellins has recently appeared (5). '
Presented, in part, before the joint meeting of southern and northern divisions of the Pacific Southwest Association of Chemistry Teachers held s t the University of Cslifonia.at Los Angeles, Febmary 23, 1957. 42
EFFECTS ON PLANTS
Flowering plants may respond to gibberellin treatment in a number of interesting ways. An acceleration of growth has been observed in a wide variety of plants in addition to rice including wheat, oats, clover, barley, buckwheat, maize, peas, cucumber, tomato, onion, lettuce, cabbage, soybeans and others. The stem and leaf parts of treated plants may become several times longer than those of non-treated control plants. The magnitude of the response depends on the species and the conditions of application. Root growth may be stimulated, though this effect is neither pronounced nor general. Under appropriate conditions these elongation effects may be accompanied by an increased dry weight. These changes are caused by an increase in either the size of the cells or the number of cells or both. Pronounced growth responses have been observed with certain dwarf strains. Brian et al., in 1954 reported that a dwarf strain of beans responded to treatment with gibberellins to give the climbing or "pole" habit of growth rather than its characteristic bushy habit (6). Brian and Hemming also reported in 1955 that some dwarf varieties of peas responded to gibberellin treatment by growing a t a rate similar to that of tall varieties (7). The tall varieties on the other hand showed little or no response to the same treatment. Phinney has tested growth responses of ten different single-gene dwarf mutants of maize to gibberellins (8, 9). Without treatment, the mutants grow to a height of 10Yo-50Yo that of normal plants. The mutants appear bushy because of the relatively short leaves and short distances between the points of attachment of the leaves. When solutions of gibberellins were applied to the uppermost leaves throughout the growing period, five of these mutants responded by giving a growth rate and appearance essentially indistinguishable from normal plants which received a similar treatment (see Fig. 1). The other five mutants showed little or no response to this treatment (see Fig. 2). No other substances tested, including many known plant growth regulators, increased the rate or extent of growth of these mutants. Treatment with gibberellins can also induce some plants to bolt and flower under environmental conditions which would not normally allow flower production. The biennial henbane, Hyocyamus niger, normally requires a cold treatment followed by long days in JOURNAL OF CHEMICAL EDUCATION
Figure 1.
Response of Maize Mutant Drarl.1 to Gibberellin Treatment
From left t o right, rmrwnl plants n l i u l l irrrivod no treatment, normal ldhnts treated with a total of 250 mirroarrma of pibberellie acid, dcuorf-2 "bnts which received no treatment and dalorf-I plants treated with 250 ,nieroerams of gibberellic heid.
order t o flower. However, Lang has de~nonstratedthat this species can be caused to flower after a daily treatment with from 2 to 10 micrograms of gibberellic acid with no special temperature or light treatment (10). This has since been verified for other plants that nonnally require eit,her a rold t,reat,mentor long days t o flower. Among other interesting examples of physiological responses in plants elicited hy gibberellins are the stimulation of frnit set in tomatoes (If), breaking of potato tuber dormancy (12), the promotion of germination in the dark of lettuce seed that normally require light for germination (13), the reversal of light inhibition of pea stem grovth (I/,) and tomato growth (15). Research in this area is intense with new findings being reported each month. The spectrum of physiological responses t o gihherellilis does not seem t o resemble that of any other group of plant growth regulators. As yet, there has been very little information on which t o base an explanation of the mechanism of action of the gibberellins or to correlate t,he various types of physiological responses ohserved. At least three different compounds which possess these biological properties have been isolated in crystalline form from the culture filtrates of Gibberella fujikuroi. Stodola, et al., fractionated their material into two components (16). Then Sumiki, et al., showed that the material with which they had been working was a mixture of three closely related compounds (17). Cross, et al., on the other hand apparently obtained only a singlecompound (18). Theidentity heb e e n compounds isolated by the different laboratories has not been fully established; however, the names and probable relationships hetween them are summariaed in Table 1. The general structural features of gibberellic acid seem well established from the studies of Cross. et al.. on the physiral and chemical properties of the acid and a number of its degradation produrts (19). O w of the strurtures proposed hy them for gihherellir VOLUME 35, NO. 1, JANUARY, 1958
Figvrr 2.
Laok of Response of Meire Mutant "Dominant Dwarf" t o
Gibbamllin Treatment From lcft to right, normrl &~nfa which received n o treatment, nornml [rlants treatcd with 200 mierograrna of gibberellic acid, m u t a n t plants whioh received no rrestment,, and m u t n n t ~ b n t trentcd s with a total of 200 mioroerhlna of gibberellic add.
TABLE 1 Gibberellins Isolated from Cibberellm fujikuroi Takahashi Empirical ,fomzlla
el a/. (17)
CLBHWO~Gihherellin At C,.H& Gihherellin A* C , Q H ? ~ & Gihberdlin A3
Fisurr 3.
Slodolo ct al.
(18)
Cross el al. (18)
Gihhercllin A Gihhercllin X
Gihberellie acid
Proporad Structure tor Gibbemllis Acid (191
:wid is shown in Figure 3. The greatest unrertainty concerns the positions of attachment of the lactone and the other functional groups in ring A. It seems a likely possibility that gibberellin A1 and gibberellin Az are dihydro- and tetrahydro- derivat,ives, respectively, of gibberellic acid. I n all the cases cited above, it should be horne in mind that the physiological response vns elicited in flowering plants by a natural product derived from a fungus. Phinney demonstrated in his work with single-gene dwarf mutants of maiae that five of t,he mutants responded t o gibberellin treatment t o give growth indistinguishable from that of normal plants. The gibberellins were the only compounds tested which gave this response. The followi~lgrationalization of these results was adopted a s a working hypothesis. The gibberellins or some materials closely related to them serve as natural growth regulators in maize. I n the dwarf mutants, the production of these regulators has in some manner been blocked. Thus, when an external supply of gibberellins is provided, the mutants are ahle to grow as rapidly as do the normal plants
which produce their own supply of gibberellins. The work described below was undertaken in an attempt to answer this question of whether gibberellins, or closely related materials, are naturally occurring growth regulators in maize and possibly other flowering plants. An account of this work has been published elsewhere (9). BIOASSAY FOR GIBBERELLIN DETECTION
It has been essential to have some means for the detection of gibberellins. A bioassay was developed h a ~ - don the growth response in the seedling stage of one of the dwarf mutants of maize, dwarf-1. This mutant was selected because of the specificity and sensitivity to applied gibberellins. The growth response to 0.0001 microgram of gibberellic acid can be detected under ideal conditions. The solution to be tested is applied as a small drop to the first unfolding seedling leaf a t the time of its emergence from the coleoptile. Following treatment, the seedling is allowed to grow until the first leaf sheath has reached its final length, a period from three t o five days following application. A record of response is obtained by measuring to the nearest millimeter the length of this sheath. With appropriate modifications, this technique can be made the basis of a quantitative bioassay for gibberellins. Preparations from a number of flowering plants have been surveyed for the presence of gibberellin-like substances with this bioassay as a guide. A group of extracts of maize plants and maize tissues was tested first, and an ethyl ether extract of maize seed in the milk stage was found to give a growth response in the mutant resembling that of the gibberellins. This information supported the idea that gibberellins may be naturally occurring growth regulators in maize and prompted the investigation of fruits or seeds from other species. In general the material was soaked in a 1:1 mixture of acetone and water, the solvent evaporated from the filtered extract, and the residue applied in a small volume of water to the test plant. I n some cases the watery endosperm from the seed was applied directly to the test plant. To date the seeds or fruits of forty-three species have been tested in this manner, and preparations from nineteen of these representing fifteen genera and seven families have given a positive response (see Fig. 4 for a typical positive response). It is not known whether seed giving inactive extracts lacked gibberellin-like substances, or whether the conditions of extraction and assay were insufficient to reveal their presence, or whether inhibiting substanres were also present in the
%pons. of Dworf-7 Trratment urith Gibwith Extrast of Bean
From left to right, a seedling which was n ot treated, a scedling treated with 10 miorog'ams of a mirture of eibbadlin At sad ribberellie acid, and s seedling treated with an extract of bean seed.
-
extract. Active extracts have been obtained from the seeds or fruits of beans, maize, peas, lupine, plums, apricots, peaches, wild buckeye, avocado, wild cucumber and wild tobacco. Preparations from six of these, namely avocado, beaus, wild cucumber, wild buckeye, lupine, and peas, have been applied to each of the ten dwarf mutants of maize with the result that the five mutants which responded to the gibberellins also responded to the plant extracts, whereas the five mutants which did not respond to the gibberellins likewise did not respond to the plant extracts. An extract of wild cucumber endosperm has been shown recently by Lang to duplicate the gibberellin effect on flowering in Hyocyamus niger (20). Radley has reported the presence of a gibberellin-like factor in alcoholic extracts of pea seedling shoots ($I), and Lona has presented evidence for the presence of a gibberellin-like factor in Brassica napus L. (22). All these results indicate the widespread occurrence of substances in flowering plants with gibberellin-like biological properties. CHEMICAL PROPERTIES OF GIBBERELLIN-LIKE SUBSTANCES
It mill be of interest to learn whether the active components of plant extracts resemble gibberellins in chemical properties as well as biological properties. Filter paper chromatography has proved useful in comparing the various active materials. Gibberellic acid was detected on developed chromatograms by means of the characteristic blue fluorescence produced when the paper was soaked in concentrated sulfuric acid and exposed to ultraviolet light. The positions on the chromatogram of gibberellin A*, gibberellin Az, and the active components of the plant extracts were determined by cutting the strips into measured zones, eluting each of these separately, and assaying the eluates. R,values were calculated as the ratio of the distance the active component had moved from the origin to that which the solvent had moved. The results of a comparison in four solvent systems are summarized in Table 2. An inspection of these data reveals differences among the active materials. The three gibberellins derived from the fungus could not be readily distinguished from one another in any of the solvent systems. The active factor from beans gave TABLE 2 Chromatographic Comparison of Active Plant Extracts and the Gibberellins Rr in solvat s y s l a Material tested A B C D Gibberellic acid 0.25 0.58 0.85 0.74 Gibberellin At 0.24 0.55 0.89 0.72 Gibberellin A2 0.29 0.61 0.87 0.73 Bean extract 0.27 0.65 0.85 0.76 Pea extract 0.43 0.65 0.91 0.73 Buckeye extract 0.43 0.66 0.86 0.74 Wild cucumber extract 0.51 0.7i 0.87 .. Lupine extract 0.11 0.48 0.67 .. Rivalues are the average of two or more determinationrr which agreed within 0.05. Solvents: A, the upper phase of a mixture of 3 volumes of n-butanol and 1 volume of 1.5 N ammonium hydroxide; B , the upper phase of a mixture of 35 volumes of pyridine, 35 volumes of n-amyl alcohol, and 30 volumes of water: C, the upper phase of s, mixture of 95 volumes of n-butanol, 5 volumes of glacial acetic aeid, and 30 volumes of water; D, a solution of 8 volumes of ethanol and 2 volumes of 3 N ammonium hydroxide.
JOURNAL OF CHEMICAL EDUCATION
R, values similar to the known gibberellins in three of the solvent systems, but gave a consistently higher value in solvent system B. The active materials from peas and buckeyes gave simiiar Rt values which differed from other materials tested. And the active materials from wild cucumber and lupine each gave a distinctive pattern. Thus, it would seem that none of these extracts has as its active component a substance chemically identical with the known gibberellins, and, furthermore, there may be a family of substances with gibberellin-like activity in higher plants. It was also noted that none of the active zones on chromatograms of the plant extracts gave a positive indication of gibberellic acid in the sulfuric acid fluorescence test, of indole compounds in the Ehrlich's aldehyde test, or of leucoanthocyauins or related materials in the vanillin test. I n order to learn the chemical nature of these active substances from plant extracts it will be necessary, of course, to isolate them in a pure state in sufficient quantities for chemical studies. This is made difficult by the apparently low concentrations in which they occur in plant materials. For example, the wild cucumber, which is one of the best sources found t o date, has an amount of gibberellin-lie activity equivalent to about one milligram of gibberellic acid per four bushels of fruit. Efforts have centered on isolating the materials from wild cucumber and bean seed. The most useful approach found t o date utilizes the techniques of charcoal adsorption, chromatography on silicic acid and charcoal and counter current distribution. Highly purified materials (more than a thousand times more active than the original concentrate) have been obtained, but as yet no products of a crystalline nature or proved purity have been isolated. Considerable effort is being devoted t o processing large amounts of plant material for these substances, and chemical studies must await the successful completion of this work. Although there remains much to be done, it seems safe to conclude that substances with gibberellin-like biological properties may be found as natural components of many, if not all, flowering plants. This fact, coupled with the knowledge of the effects which gibberellins have on many plant processes such as
VOLUME 35, NO. 1, JANUARY, 1958
growth and flowering, points to these substances as natural regulators of these processes. Thus, it seems likely that further study of the nature and action of gibberellins will advance our understanding of plant physiology and, a t the same time, may provide us with materials and methods which can be used t o economic advantage in controlling the development of commercial crops. LITERATURE CITED (1) YABUTA, T., AND Y. SUMIKI, J . Agr. Chem. Soc. Japan, 14, 1526 (1938). (2) MITCHELL, J. E., AND C. R. ANGEL, Phytopalhology, 40, 872 (1950). (3) STODOLA, F. H., K. B. RAPER,I).I. FENNELL, H. T. CON-
C. T. LANGFORD, AND R. W. JACKSON, WAY,V. E. SOHNS, A ~ c hB. i o c h . Biophw., 54,240 (1955). (4) Bonnow, A., P. W. BRIAN,V. E. CHESTER, P. J. CURTIS, E. G. JEFFRIES,P. B. H. G. HEWING,C. HENEHAN, LLOYD, I. S. NIXON,G. L. F. NORRIS,AND M. RADLEY, J . 8.5. Food Agr., 6 , 340 (1955). (5) STOWE, BRUCEB., AND T. YAMAKI, Ann. Re". Plant Phusiol., 8, 181 (1957). (6) BRIAN,P. W., G. W. ELSON,H. G. HEMMING, AND M. J . Sei. Food Agr., 5, 602 (1954). RADLEY, (7) BRIAN,P. W., AND H. G. HEMMING, Physiol. Plantarum, 8, 669 (1955). (8) PHINNEY, B. O.,Proc. Nat. Acad. Sci. U.S., 42,185 (1956). (9) PHINNEY, B. O., C. A,, WEST, MARYRITEEL,AND P. M. NEELY,Proc. Nat. A d . Sei. U.S., 43, 398 (1957). (10) LANG,ANTON, Naturwissensehaften, 12, 284 (1956). (11) W I ~ RS. , H., M. J. BUKOVAC, H. M. SELL,AND L. E. WELLER, Plant Physiol., 32,39 (1957). AND M. RADLEY, Phl~giol. (12) BRIAN,P. W., H. G. HEMMING, Plantalum, 8,899 (1955). J. A. GOSS,AND D. E. SMITH,Science, 125, (13) KAHN,ALBERT, 645 (1957). (14) LOCKHART, JAMES,PTDC.Nat. Acad. Sei. U . S., 42, 841 (1956). (15) LIVERMAN, J. L., AND S. P. JOHNSON, Sciace, 125, 1086 (1957). (16) STODOLA, F. H., G . E. N. NELSON,AND DEANJ. SPENCE, Arch. Biochem. Biophys., 66, 438 (1957). (17) TAIUHASHI,N., H. K I T A M ~ A. A ,KAWARADA, Y. SETA,M. (18) (19) (20) (21) (22)
TAKAI,S. Tnwrma, AND Y. S ~ I K IJ,. Agr. Chem. Soe. Japan, 19,267 (1955). CROSS,B. E., J . Chem. Soe., 1954, 4670. CROSS, B. E., JOHNF. GROVE, J. MACMILLAN, AND T. P. C. Chem. & Ind. ( L o n d a ) , 1956, 954. MULHOLLAND, LANG,ANTON, unpublished observations. RADLEY, MARGARET, Natum, 178, 1070 (1956). LONA,FAUSTO, L'Ateneo P a r m e , 28, 111 (1957).
