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First Symposium on Chemical-Biological Correlation, May 26-27, 1950 (1951)

Chapter: Structure-Activity Relationships in Plant Growth-Regulators

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Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 46
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 47
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 48
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 49
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 50
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 51
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 52
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 53
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 54
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 55
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 56
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 57
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 58
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 59
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 60
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 61
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 62
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 63
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 64
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 65
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 66
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 67
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 68
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 69
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 70
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 71
Suggested Citation:"Structure-Activity Relationships in Plant Growth-Regulators." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 72

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STRUCTURE-ACTIVITY RELATIONSHIPS IN PLANT GROWTH-REGULATORS A. G. Norman and R. L. Weintraub Chemical Corps Biological Laboratories Camp Detrick, Frederick, Maryland CONTENTS Page I. Introduction 46 II. Limitations of Existing Data 46 III. Stimulation of Cell Elongation 47 IV. Initiation of Roots 54 V. Induction of Parthenocarpy 57 VI. Modification of Organs 58 VII. Control of Abscission 6Z VIII. Control of Bud Development 64 IX. Repression of Root Elongation and of Seed Germination . . 65 X. General Discussion 68 Literature Cited 70

46 I. INTRODUCTION A large number of organic compounds are known which have the property, in relatively small amounts, of influencing the mode or rate of the growth and developmental processes in plants. Such compounds, the majority of which have not as yet been demonstrated to occur naturally in higher plants, may be termed growth-regulators. The growth responses may be identical with, or similar to, those normally taking place or may be of a type not found in the intact healthy plant. Responses in the first category include acceleration of rate of cell elongation, acceleration or retardation of abscission, induction of parthenocarpy, stimulation or inhibition of development of dormant buds, and development of adventitious roots. Among the second type of responses may be mentioned production of galls and tumors, and modifications of leaves, flowers, and other organs. More or less profound changes in composition and metabolic processes doubt- less cause, accompany, and result from these morphological changes, but these have been studied much less extensively. Although the relationships between molecular structures and physiological potency of plant growth-regulators have been under consideration for some time, the present status is by no means satisfactory. Reference should be made at this point to a number of earlier publications dealing with certain of the topics to be treated'6, 21, 28, 29, 39, 44 No attempt will be made in this review to catalog completely all the substances which have been stated to possess growth-regulatory activity or even to mention all the known types of responses. One notable omission is that of herbicidal activity, the study of which, although of great practical interest, has not yet reached the stage of profitable discussion in the present connection. II. LIMITATIONS OF EXISTING DATA Attempts to deduce general principles from the data now available are so greatly complicated by a variety of circumstances that present postulates must be regarded as provisional. Certain compounds possess activity in many of the types of response enumerated above, thus lending weight to the view that these responses, although not otherwise demonstrated to be related, may be diverse end-effects of a common fundamental reaction. On the other hand, some compounds exhibit high activity in one type of response but none at all in others. Regrettably, there has too frequently been a tendency to overlook this heterogeneity of behavior and to speak of structural requirements of growth-regulators in general. For this reason it appears advisable to discuss the structural requirements separately for each type of activity. A further complication is introduced by evidence of more or less marked differences in sensitivity or responsiveness of similar organs of various species and also, for that matter, by the same organ at different stages of development. At present it is not clear to what extent such specificity may be due to fundamental differences in tissue or cellular reactivity and to what extent to secondary properties which may influence absorption, trans location, or destruction of chemicals applied. Apart from the variability inherent in biological material, much of the uncertainty of current data is attributable to inadequate techniques. In relatively few cases have extensive series of compounds been compared by carefully standardized tests. A number of serious

47 inconsistencies have been reported by different investigators using a presumably standard technique, and even by the same worker in experiments performed at different times. Many of the published results are to be regarded as semi-quantitative at best; in numerous instances it has been determined only whether a compound is active or inactive at arbitrarily selected dosages. In development of test methods, conditions are commonly adjusted so as to result in a maximal response to a particular standard compound, frequently indole-3-acetic acid or 2, 4-dichloro- phenoxyacetic acid. The time of exposure or other experimental conditions may not be optimal for other substances which may enter, move, react, or be inactivated at appreciably different rates. In consequence, the relative activity of any substance, compared to another substance as standard, varies according to the method of assay. This situation, which was clearly recognized in the early investigations of structure and activity, has not been sufficiently emphasized in some of the later discussions of the subject. Only recently have attempts been made to devise tests so controlled as to permit expression of results in more or less absolute terms such as the dose of half-maximal response (ED 50)5. 13, 19 It is to be remarked that the responses under consideration include both stimulatory and repressive growth effects. In the normal economy of the plant both types play equally indispens- able roles. From the experimental standpoint, however, much less ambiguity attaches to the study of stimulatory or inductive responses. Repression of growth may be brought about by almost any material in sufficiently high concentration, including even the essential nutrients and metabolites. Current experimental techniques are almost exclusively restricted to measurement of gross changes in complex organs so that the question arises to what degree the repressive or inhibitory activity of any particular compound may be regarded as specific for the response under investigation. III. STIMULATION OF CELL ELONGATION The opinion is widely held that stimulation of cell elongation represents the simplest morphological effect of growth-regulators, and in consequence the structural requirements for this response have been more extensively studied than for any others. Cell elongation is a process which is an important component of plant growth, and which is involved also in tropistic and etiolation responses. Compounds, whether of natural origin or not, which stimulate the elongation of cells have been termed "auxins" and although broader definitions have at times been given, the term will be used in this paper in this restricted sense. Compounds having auxin-activity, therefore, are those which can be shown to induce cell elongation in standard tests. The available test methods depend upon measurements of overall growth of organs, or segments of organs, comprising a variety of tissues of different cell types. Such test objects, although less complex than the entire plant, nevertheless are far from simple, and part of the difficulty in interpretation and correlation of experimental results arises from the multiplicity of physiological processes involved Some advantage might be derived from a test utilizing single tissues or cells but none such has yet been devised. Four principal types of assay methods have been utilized in investigating activity of this type: (a) Curvature or elongation of decapitated, but rooted, cereal coleoptiles growing in air. This comprises the classical A vena coleoptile curvature test, and its various modifications. (b) Elongation of excised segments of etiolated coleoptiles or internodes immersed in solution.

(c) Curvature of slit halves of etiolated pea internodes immersed in solution. (d) Curvature or elongation of stems, petioles or coleoptiles of green or etiolated intact plants to which small volumes of test material are applied at the responsive sites. The physiological activity of any substances is a function not only of its effectiveness once it has gained access to the interior of the cell (primary activity) but also of various secondary properties such as rates of penetration into the test organ, transport through it, and ease of conversion within the tissue either to more active or less active form than the starting material. In methods (b), (c), and (d) the test material is applied in closer proximity to the reactive cells than in method (a) so that the necessity of internal movement may be presumed to be less. A number of compounds which exhibit little or no potency by method (a) are active when tested by one or more of the other techniques, presumably owing to this factor, whence the latter tests have been regarded as superior for the demonstration of primary ..activity. For the present purpose a substance active in any of these types of test will be regarded as possessing primary activity. There is reason to suspect, however, that all the available methods fall far short of the ideal test in which the intrinsic activity would be unobscured by factors such as entry into the cell. Some hundreds of compounds have been investigated by these methods and a substantial number found to exhibit activity. A question of paramount interest is whether all active compounds possess identical primary activity modified to the observed potency by secondary differences in properties. Such a view has been advocated on the basis of rather limited experimental evidence3. 3V but in the light of more recent results does not appear tenable. The first systematic studies 10. 17 of the relationship of structure to auxin-type activity were published in 1935 and soon thereafter Koepfli, Thimann, and Went'" were able, on the basis of results obtained with the pea test, to codify the multiplicity of active substances by postulating the following minimum structural requirements for primary activity: (1) a ring system as nucleus ;'.••') a double bond in this ring (3) a side chain (4) a carboxyl group (or a structure readily converted to a carboxyl) on this side chain at least one carbon atom removed from the ring (5) a particular space relationship between the ring and the carboxyl group More recently these requirements have been restated as "an unsaturated ring system, with a side chain, adjacent to the ring double bond, of at least two carbon atoms ending in a carboxyl group"38 Although it is true that the majority of active compounds which have been discovered in the past fifteen years conform to these principles, there are known also a number of apparent exceptions. Conversely, the existence of some inactive compounds which also possess the stipulated structural features provides evidence that these have been incompletely defined. In the following paragraphs each of the putative requisite features will be taken up separately. The ring system. Among compounds of demonstrated activity are found all the major types of rings: carbocyclic and heterocyclic, aromatic and alicyclic, simple and fused (Tables I, II). Active compounds are known with the following rings: Cyclopentene, cyclohexene, benzene, naphthalene, anthracene, acenaphthene, fluorene, indene, indane, benzofuran, indole, coumar^n, thianaphthene. The activity of a compound containing one of these rings may be strongly influenced by the nature and position of attachment, both of the acidic side chain and of other ring substituents. In no ring series has a sufficient number of compounds been tested fully to determine these influences so that it appears premature to attempt to compare activities of various rings in general. In many of the ring series with active compounds there are also inactive members. Hence, in reaching conclusions as to the activity of any ring in general, it is necessary that a large number of derivatives be examined. The finding of one or a few inactive compounds, as e.g. certain of the acetic acid derivatives of pyridine, pyrrole, furan, thiazole, uracil, iminazole, carbazole, and dibenzofuran, does not furnish an adequate basis for

49 TABLE I COMPOUNDS EXHIBITING HIGH AUXIN ACTIVITY CHOHCH2CHOHCHOHCOOH CH3 CH3 Auxin-a (Auxentriolic acid) CH2CH2CH2COOH Y-(Indole-3)-butyric acid CHOHCH2COCH2COOH CH3 CH3 Auxin-b (Auxenolonic acid) CH2COOH H Indole-3-acetic acid CH3 CHCOOH a-(Indole-3)-propionic acid CH2CH2COOH H p-(Indole-3)-propionic acid CH2CH2COOCH CH2CH2CH2CH2COOH H ^-Indole-3-valeric acid ,N jCH2COOH 5-Methylindole-3-acetic acid CH = CH l COOH Allo (cis)-cinnamic acid CH2COOH GO Naphthalene-1-acetic acid CO CH2COOH Naphthalene-2-acetic acid H Isopropyl p-(indole-3)-propionate

50 TABLE I (Cont.) cool CH2COOH Anthraceneacetic acid Cl X\OCH2 ,COOH 4 *"hlorophenoxyacetic acid OCH2COOH 2, 4-Dichlorophenoxyacetic acid |CH2COSH 2-Methyl-4-chlorophenyl-thioacetic acid ClOCH2COOH 2,4, 5-Trichlorophenoxyacetic acid CH3 nCHCOOH 2, 4-Dichlorophenyl-o-propionic acid 2, 4-Dichlorophenoxyacetanilide Cl 2, 4-Dichlorophenoxy) -ethylamine TABLE II REPRESENTATIVES OF TYPES OF RING COMPOUNDS ACTIVE IN STIMULATING CELL ELONGATION C CCH,COOH .iCOOH Indene -3 -acetic acid 1-Cyclohexene-1-acetic acid

TABLE II (Cont. ) HCOOH Benzofulvene carboxylic acid H2COOH H2C CH2 Acenaphthene-S-acetic acid CH2COOH Benzofuran-3-acetic acid CH2COOH -- CH2COOH Fluoreneacetic acid COOH Coumaran-3 -acetic acid CH2COOH a-1, 2, 3, 4-Tetrahydronaphthoic acid Thianaphthene- 3 -acetic acid assuming that differently substituted derivatives of these nuclei also must be without activity. On the basis of present evidence, however, it appears that compounds with simple rings are in general less active than those with fused rings, the outstanding exceptions being auxins a and b. It has been postulated that the activity of a ring depends upon the presence of reactive double bonds therein but present information is insufficient to support this view. On the one hand, high activity is exhibited by some benzene derivatives which might be expected to possess relatively unreactive unsaturated linkages, while, on the other hand, low activity is shown by a number of derivatives with highly reactive double bonds in rings such as those shown in Table II Indeed, even the requirement of the presence of a double bond in the ring adjacent to the side chain can scarcely be maintained in view of the activity of coumaran-3-acetic acid3?, a-1, 2, 3-tetrahydro- naphthoic acid , and the so-called benzofulvenecarboxylic acidZS. The carboxyl group. A considerable number of active compounds without a free carboxyl group in the side chain is known. Some of these, such as esters, amides, lactones, and nitriles may be hydrolyzed in vitro to yield a carboxyl but there is no direct evidence that these compounds become active in vivo only after hydrolysis, and indeed there is some evidence that this is not the case. If only the compound with free carboxyl were active it would be expected that the acid and its hydrolyzable relative would show parallel activity-concentration

52 curves. In the instance of naphthalene-1-acetic acid and its amide these curves are quite different; furthermore, no trace of ammonia could be detected at optimal concentrations of the amideZS. Although in a number of examples esters and amides are less active than their correspond- ing acids this is not true without exception. The amides of 2, 4-dichlorophenoxyacetic, ^-(2,4- dichlorophenoxy )-butyric, and € -(2, 4-dichlorophenoxy)-caproic acids are somewhat more active than the acids themselves". The isopropyl ester of indole-3-acetic acid has activity equal to that of the free acid and the methyl ester of 2-methylindole-3-acetic acid is considerably more active than its acid. Also the methyl and isobutyl esters of phenylacetic acid exhibit equal activity although they may be presumed to be hydrolyzed at quite different rates39. Naphthalene -1 -acetonitrile, while active in some tests, induces a much delayed response compared to the free acid'O, a result in harmony with the view that conversion of the former compound to the latter is involved. However, the possibility that some other factor, such as slow penetration, may be responsible for the delayed effect has not been excluded. The activities of indole-3-acetaldehyde and of 2, 3, 5-trichlorobenzaldehyde quite conceivably may be due to in vivo oxidation to the corresponding acids. On the other hand, in the case of p-(2, 4-dichlorophenoxy)-ethylamine, which has appreciable auxin activity, an oxidation and deamination would be required for conversion to an acid. The same would be true of 3-(indole)-ethylamine which exhibits activity only after prolonged contact with the test plant^. Benzoyl oxide and benzoyl peroxide have been reported as active in a modification of the A vena test". Activity of the latter compound could not be confirmed in either the Avena or the pea tests"3 but this may have been due to its rapid inactivation. In any event, the stipulation of a carboxyl group appears too stringent, because compounds with other types of acidic functions also may possess activity. The acid grouping may be isosteric with carboxyl, as in the case of the aci-form of naphthyl- 1 -nitromethane, or not, as in potassium indoxylsulfate^. The side chain. The postulated requirement of a carbon side chain separating the acid group from the ring also cannot be maintained. Compounds in which a carbon in the side chain is replaced by a heteroatom, such as oxygen, nitrogen or sulfur have been shown to possess activity. The most obvious example is 2, 4-dichlorophenoxyacetic acid. Others are N-(2,4- dichlorophenyl)-glycine, which has been tested in our laboratory, and S-(2-methyl74-chloro)- thioglycollic acidZS. Furthermore, there are now known several active substances in which the carboxyl group is attached directly to the ring. These include substituted benzoic acids, such as 2-bromo-3- nitrobenzoic acid, 2-bromo-3, 5-dichlorobenzoic acid, and 2, 3, 5-trichlorobenzoic acid from among the many benzoic acids studied at Camp Detrick, 2, 3, 6-trichlorobenzoic acidZ, a-naph- thoic acid and a-l , 2, 3, 4-tetrahydronaphthoic acid3'. Nevertheless, the character of the side chain appears to be of considerable importance. Thus, in the pea test the activity of phenylacetic acid is not appreciably diminished by substitution of a single methyl group in the a-position whereas activity is lost on introduction of a single phenyl or hydroxymethyl group, or of two methyl groups, or one methyl and one hydroxyl at this locus. Similarly, the primary activity of indole-3-propionic acid is not affected by introduction at the a-position of a methyl group but is completely nullified by carbomethyl (indole-3-succinic acid). This cannot be attributed merely to the presence of two carboxyls since other dicarboxylic acids (m-phenylenediacetic diethyl ester and indylene-1, 3-diacetic acid) are active. The carboxyl-bearing side chain may bear other functional groups without complete loss of activity as in ej-aminophenylglyoxylic acid and indole-3-pyruvic acid. Tryptophane gives a delayed response suggesting need of transformation in vivo. Activity is not necessarily nullified by the presence of a double bond in the side chain, either adjacent to the ring as in 1, 2, 3, 4-tetrahydronaphthylidene-l-acetic and benzofulvenecarboxylic acids, or elsewhere, as in c_is-cinnamic acid.

53 The position of attachment of the side-chain in an unsymmetrical ring system may be of significance. Thus, appreciable differences of activity appear to exist between the members of the following pairs: coumaran-3-acetic > coumaran-2-acetic ; 2-naphthoxyacetic > 1-naphthoxy- acetic; naphthalene-1-acetic > naphthalene-2-acetic; p-naphthalene-2-propionic > p-naphthalene- 1 -propionic. Length of the side chain also may be of importance. In the homologous series of omega- substituted phenyl aliphatic acids from phenylacetic to phenylvaleric only the first member is active. On the other hand, in the similar series from indole-3-acetic to indole -3 -valeric all the members exhibit approximately equal activity. In the series from 2, 4-dichlorophenoxyacetic to 2, 4-dichlorophenoxycaprylic a clear-cut periodic effect of side chain length is apparent. The compounds with side chains containing an even number of carbon atoms are active, whereas the alternating odd-numbered members are inactive; the same effect is found among the amides of this series. Similarly, p-(2-naphthoxy)-propionic acid is inactive although the acetic and butyric homologs are active. A possible explanation of this periodicity in the 2, 4-dichlorophenoxy series is to be found in the suggestion that p-oxidation of the acidic side chain precedes functioning of the higher homologs. In this way the active acetic homolog could be produced only from the even-numbered members, while the odd-numbered compounds would lead to unstable aryl carbonates^ Ring substituents. Relatively few derivatives of active compounds in the indole series have been available for assessing the role of substitution into the ring. Introduction of methoxyl at positions 5, 6, or 7 in indole-3-propionic acid leads to loss of activity, whereas the corresponding derivatives of indole-3-acetic acid remain active*-'°. 1-Methyl-, 2-methyl-, 5- methyl-, and 2, 5-dimethylindole-3-acetic acids are active, whereas the 2-ethyl derivative is not. Introduction of one or two nitro groups into phenylacetic acid abolishes activity. Conversely, the activity of phenoxyacetic acid is markedly augmented by appropriate substitution of chlorine, bromine, or nitro into the ring. 3-Nitrophenoxyacetic acid is active butthe 2- and 4-isomers are not. The activity of phenoxyacetic acid is enhanced also by a single halogen atom in the 3 or 4 position but not in the 2 position. However, introduction of two chlorine atoms in positions 2 and 4 leads to even greater increase in activity. 2, 4, 5-Trichlorophenoxyacetic has high activity, whereas the corresponding 2, 4, 6-compound has virtually none. On the other hand, 2, 3, 6-trichlorobenzoic acid has high activity, and 2,4, 6-trimethylphenoxyacetic acid also is active2,20. From the information presently available, it does not appear possible to conclude generally that any particular position in any ring series must be substituted or unsubstituted in order to permit activity. Spatial configuration. The spatial configuration of the molecule may be of great importance in determining activity. Two examples of the influence of optical isomerism are given by the a-(2, 4-dichlorophenoxy)-propionic acids of which the D form has twice the activity of the racemic mixture, indicating virtually no activity for the L formZ8 and by the 1,2, 3,4- tetrahydronaphthoic acids of which the (-) form is much more active than the ( + ) form (Veldstra, cited by Thimann28) The classical example of the importance of geometrical isomerism is that of the cinnamic acids, of which cis form is active, whereas the trans isomer is inactive. More recently, two additional pairs of isomers have been discovered - the 1,2, 3, 4-tetrahydro- naphthylideneacetic acids and the naphthaleneacrylic acids - in each of which one form is active and the other not. The importance of spatial configuration, together with other considerations, have lead Veldstra^' to the following generalization of the structural requirements for auxin activity: (1) A basal ring system with high surface activity, (2) a carboxyl group (or its dipole) in a very definite spatial position with respect to this ring system. The necessary spatial position is one in which the polar group is situated outside the plane of the ring system.

The evidence which has been assembled in support of the requirement of possession of high surface activity by the ring system cannot be reviewed here . "' '' . ". It has been suggest- ed that growth-regulatory activity is related to the relative lipophilicity (due to the surface-active properties of the ring) and hydrophilicity (contributed by the carboxyl group) of the molecule. A number of otherwise puzzling cases may be explicable in terms of the spatial relation- ships of ring and side chain. One example is that of the three isomeric mononitrophenoxyacetic acids of which only the meta isomer has activity. This compound is incapable of mesomerism whereas the ortho and para isomers are able to resonate in equilibrium with respective quinoid forms in which the carboxyl group is claimed to be more restricted to the plane of the ring. Another example is that of a-l, 2, 3, 4-tetrahydronaphthoic acid which, in contrast to naphthoic acid, is active. Models indicate the carboxyl group of the former to be held outside the. plane of the ring, whereas this is not true of the latter. On the other hand there are many compounds, the activity of which have not yet been satisfactorily explained on the basis of Veldstra's postulates. Whatever the ultimate fate of this concept, it has the merit of providing a fresh working hypothesis for further experimentation. Reference should be made at this point to a group of compounds which possess none of the above-postulated requisite structural characteristics but nevertheless include some of the most potent growth-regulatory substances known. These are carbon monoxide, the unsaturated hydrocarbons ethylene, propylene, butylene, and acetylene, and the halogenated hydrocarbons ethyl bromide, ethyl iodide, and propyl chloride. These gases, in low concentration, are capable of inducing differential rates of growth of upper and lower sides of petioles which result in growth curvatures of the organs. Zthylene is inactive in the pea test although it has been shown to accelerate the growth rate of certain other seedlings. What relation these substances may bear to the ring type of auxins is uncertain, but two points of interest may be mentioned. Firstly, unlike the great majority of the compounds previously discussed, ethylene is known to be a natural product of many kinds of plants, and there is considerable evidence that it may play an important role in plant metabolism and behavior. Thus, it appears to merit the designation phytohormone as well as, if not better than, any other known compound. Secondly, it has been shown that volatile emanations, having the physiological properties of ethylene, are produced by certain plants after treatment with growth-regulators such as indole-3-acetic acid5O. Other physiological responses to ethylene and related compounds will be referred to in later sections. IV. INITIATION OF ROOTS A striking response, induced by a large variety of compounds, is the production of roots on plant organs, such as stems, leaves, and flowers, which do not normally bear them. Such adventitious roots may result from stimulation of pre-existent root primordia or may be initiated from cells of diverse tissues. Various fragments of many species of plants show a more or less strong tendency toward production of adventitious roots without the intervention of exogenous growth-regulators, a circumstance which has long been utilized in vegetative propagation. Consequently, in testing the root-initiating activity of chemical agents conditions should be selected so as to minimize the rooting of untreated test objects. Stem cuttings have been employed almost exclusively as test objects Because of the great interest in practical application of rooting compounds much of the available information has been obtained from trials made with species of horticultural value, and relatively little attention has been given to development of standard tests. Accordingly, there is an abundant literature describing results of rooting tests with a wide variety of species. Unfortunately it is not always possible to compare the results of one worker with those of another as techniques have differed greatly and there has

55 been relatively little attempt to control influential environmental factors. With these considerations in mind, some generalizations can be made. The structural requirements for root-initiating activity appear rather less stringent than those for auxin activity. Virtually every compound which possesses any activity in cell-elongation stimulation is active also in root initiation. In addition, several substances lacking the former type of activity have been reported as possessing the latter. A list of these compounds is given in Table III. TABLE III COMPOUNDS INACTIVE IN STIMULATING CELL-ELONGATION BUT REPORTED ACTIVE IN ROOT-INITIATION Cinnamic acid Naphthalene-1-methanesulfonic acid Diphenylacetic acid Uracil-4-acetic acid Nitrocinnamic acid N-Carbethoxymethylquinolinium chloride Coumarin 4-Methylthiarole-5-acetic acid Phenylacetamide Desoxycinchotenine Desoxycinchotenidine In Table IV are included compounds alleged to be active for root initiation but not heretofore tested for auxin activity. It should be pointed out that there has been no published confirmation of some of these reports. TABLE IV COMPOUNDS REPORTED ACTIVE IN ROOT-INITIATION BUT NOT TESTED FOR CELL-STIMULATION ACTIVITY Tetrahydrofurfuryl alcohol 3, 4-Dihydronaphthalene-l -acetic acid Calcium furoate 1,2, 3,4-Tetrahydronaphthalene-6-acetamide Nicotine Sodium tetralolacetate Sodium naphthol-4-sulfonate Aminophenylacetic acid Naphthalene tetrachloride £-Phenylenediamine L-Proline 8-Hydroxyphenylthiocarbamate Anthracene 8-Hydroxyphenyl-2-iminothiocarbonate Anthraquinone-p-sulfonic acid 6, 8-Dihydroxyphenyl-2-iminothiocarbonate Anthranilic acid Naphthalene-1-acetamide Salicylideneacetamide Naphthalene -1 -thioacetamide

TABLE IV (Cont. ) a-(Naphthalene-l)-propionic acid P-(Naphthalene-1 )-propionic acid Y-(Naphthalene-1)-butyric acid V-{Naphthalene-2)-butyric acid i-(Naphthalene-l)-valeric acid C-Naphthalene-1 )-hexoic acid Naphthalenediacetic acid Naphthalene-1 -acetylglycine 2-Methylnaphthalene-1-acetic acid 2 -Methylnaphthalene - 1 -acetamide 4-Methylnaphthalene-1-acetic acid 4-Methylnaphthalene-1 -acetamide Vanillic acid Vanillin Sulfanilamide Piperonal Methoxysalicylaldehyde a-(2 -Chlorophenoxy) - propiomr acid a-(3-Chlorophenoxy)-propionic acid a-(2-Chlorophenoxy)-n-butyric acid a-(3-Chlorophenoxy)-n-butyric acid o-(4-Chlorophenoxy)-propionic acid a-(4-Chlorophenoxy)-n-butyric acid a-(2, 4-Dichlorophenoxy)-propionic acid a-(2. 4-Dichlorophenoxy)-n-butyric acid q-(2,4-Dichlorophenoxv)-i«o-valeric acid 2,4-Dibromophenoxyacetic acid a-(2, 4-Dibromophenoxy)-propionic acid a-(2,4-Dibromophenoxy)-n-butyric acid 2, 4-Diiodophenoxyacetic acid 2-Iodophenoxyacetic acid 3-Aminophrnoxyacetic acid 4-Aminophenoxyacetic acid 2-Nitrophenoxyacetic acid 3-Nitrophenoxyacetic acid a - (Phenoxy)-propionic acid a-(Phenoxy)-n-butyric acid a-(2-Methylphenoxy)-propionic acid 2-Methylphenoxyacetic acid 4-Methylphenoxyacetic acid u-(2-Methylphenoxy)-n-butyric acid Q-(4-Methylphenoxy)-propionic acid a-(4-Methylphenoxy)-n-butyric acid a-(2, 5-Dimethylphenoxy)-propionic acid o-(2, 5-Dimethylphenoxy)-g-butyric acid Pyrrole-2-acetic acid Furylpropionic acid Hydantoinpropionic acid o-(2, 4, 5-Trichlorophenoxy)-propionic acid a-(2,4, 5-Trichlorophenoxy)-n-butyric acid 2, 6-Diiodo-4-carboxyphenoxyacetic acid 2,4, 6-Triiodophenoxyacetic acid 2, 6-Dibromo-4-aminophenoxyacetic acid 2, 6-Diiodo-4-carboxyphenoxyacetic acid Dithiobiuret Salicylacetone £i3-Thiophane-2, 5-dicarboxylic acid Indole-3-acrylic acid

57 V. INDUCTION OF PARTHENOCARPY In many species of plants development of fruit from floral tissues takes place only after pollination is accomplished. It has been known for nearly half a century that the stimulating role of the pollen is attributable to a substance, or substances, contained therein and is separate from its function in fertilization. During the past decade the availability of pure compounds of demonstrated activity in other types of plant growth response has resulted in the discovery that many of these are capable also of inducing parthenocarpy - the development of fruits without pollination. Such fruits are of course seedless. Because of the practical applicability of artificial parthenocarpy, the bulk of presently available information has accumulated in much the same manner as that relating to root initiation. Attempts have been made to ascertain which compounds and formulations might be of greatest service under conditions of large-scale production of species of commercial interest, with due regard for ancillary effects of size, shape and quality of fruits and on the vegetative organs. Unfortunately, experimentation undertaken with such objectives, though of the greatest practical value, is frequently difficult of interpretation from the viewpoint of the relation between structure and activity. Only recently have quantitative tests which may be suitable for this purpose been devised'9 and virtually no results are yet available. There is considerable evidence indicating that in such studies a distinction should be made between the processes of setting and of subsequent growth of the fruit; frequently this has not been done. For these reasons it seems desirable at present only to list the compounds which have been reported as parthenocarpically active without attempting quantitative comparison of activities (Table V). It is to be noted that particular compounds may show activity on some species but not on others, or under different circumstances. TABLE V COMPOUNDS ACTIVE IN INDUCING PARTHENOCARPY 2, 5-Dichlorobenzoic acid Phenylacetic acid a-(Phenoxy)-propionic acid a-(Phenoxy)-s-butyric acid 2-Chlorophenoxyacetic acid a-(2-Chlorophenoxy)-propionic acid a-(2-Chlorophenoxy)-n-butyric acid a-(3-Chlorophenoxy)-propionic acid o-(3-Chlorophenoxy)-n-butyric acid 4-Chlorophenoxyacetic acid a-(4-Chlorophenoxy)-propionic acid a-(4-Chlorophenoxy)-n-butyric acid 2-Methylphencocyacetic acid a-(2-Methylphenoxy)-propionic acid 2, 4-Dichlorophenoxyacetic acid o-(2, 4-Dichlorophenoxy)-propionic acid a-(2, 4-Dichlorophenoxy)-fl-butyric acid 2, 5-Dichlorophenoxyacetic acid 2,4-Dimethylphenoxyacetic acid a-(2, 4-Dimethylphenoxy)-propionic acid 3,4-Dimethylphenoxyacetic acid a-(3, 4-Dimethylphenoxy)-propionic acid a-(2, 5-Dimethylphenoxy)-propionic acid a-(2, 5-Dimethylphenoxy)-n-butyric acid

58 TABLE V (Cont. ) 2, 4, 5-Trichlorophenoxyacetic acid Fluorene-4-acetic acid a-(2, 4, 5-Trichlorophenoxy)-propionic acid Phenanthrene-9-acetic acid a-(2, 4, 5-Trichlorophenoxy)-n-butyric acid Pyrrole-2-carboxylic acid 2,4, 6-Trichlorophenoxyacetic acid Pyrrole-2-acetic acid 2,4, 5-Trimethylphenoxyacetic acid Indole-3-acetic acid p-(2, 4, 6-Trichlorophenoxy)-p"-chlorodiethyl ether (5-(Indole-3)-propionic acid Naphthalene-1 -acetic acid y-(Indole-3)-n-butyric acid Naphthalene-1 -acetamide Acenaphthene Naphthalene-1-propionic acid Skatole Naphthalene-I-butyric acid Sulfanilamide p-Naphthoxyacetic acid Oestrone P-Naphthoxypropionic acid Colchicine Parthenocarpic activity, though exhibited by many substances active also in other responses, is by no means restricted to these. The structural requirements for parthenocarpic induction, like those for root initiation, appear considerably less circumscribed than in the cell- elongation or formative responses. VI. MODIFICATION OF ORGANS Certain types of compounds have been shown capable of profoundly modifying the size, shape, and texture of developing leaves. This so-called "formative" effect is due to production of closely packed, thick-walled parenchyma-like cells in place of the normal chlorophyllous mesophyll tissue and failure of lateral expansion of the leaf3S. It is of interest that many, though not all, of the useful herbicidal growth-regulators show high formative activity and also that certain of the leaf modifications may resemble those of virus-infected plants. Activity of this type was reported first for naphthoxyacetic acids'2. Subsequently, a large number of substances have been tested for formative activity by workers at the Boyce Thompson Institute25- 26,40,41,42,43,45,46,47,48,49. The use of a variety of test objects and techniques, some essentially qualitative or at best semi-quantitative, renders difficult a comprehensive comparison of the activities of all the compounds investigated. More recently, a quantitative assay method for formative activity has been devised at Camp Detrick5; ratings are made on the basis of the amount of growth-regulator which, after application to the terminal bud of a bean seedling, is required to repress the leaf expansion by 50 per cent while producing also the characteristic morphological modifications. The following discussion is based primarily

59 upon the heretofore unpublished Camp Detrick results, which are in general agreement with those of the Boyce Thompson investigations. It should be pointed out that injury of a non-formative nature is caused by many substances if applied in relatively large amounts. This sets a methodological limit to the doses that can be tested. Compounds which are rated as inactive at dosages up to this limit conceivably might exhibit activity if it were possible to test them at higher levels. With few exceptions, all the compounds so far demonstrated to possess formative activity of this type are derivatives of phenoxyacetic, naphthoxyacetic, or benzoic acid. Many closely related compounds, some of which are highly active in other types of response, are ineffective. Even among those series containing active members, details of molecular architecture play an important role. These will be discussed separately for the various series. Phenoxyacetic acid itself is inactive, and activity is not conferred by introduction into any position of the ring of a single amino, nitro, or carboxyl group. Active compounds are obtained, however, by introduction of a methyl, oximinomethyl, methoxyl, or halogen into the ring in suitable position. Of these groups the halogens exert a much greater activating influence than the others, in the order F > Cl > Br > I. Of the monochloro- and monobromo- phenoxyacetic acids, of which all the possible isomers have been examined, only those substituted in position 4 are active. On the other hand, 3- and 4-methylphenoxyacetic acids show approximately equal activity, which is, however, of a low order. Introduction of additional ring substituents into a highly active monosubstituted phenoxy- acetic acid usually tends to diminish the activity. Thus, as compared with 4-chlorophenoxy- acetic, 2, 4-dichloro is about three-fourths as active and 3,4-dichloro about one-seventh. Slight activity is shown by 2, 6-dichloro while the 2, 5- and 3, 5-isomers are inactive. So far as other dihalogenated compounds have been tested it appears that an inactivating tendency of similar magnitude is exerted by a second atom of iodine and even more strongly by fluorine or bromine. In this respect also the methyl-substituted compounds exhibit a somewhat different behavior, the 2,4-, 2, 5-, and 3, 4-dimethylphenoxyacetic acids showing activity approximately equal to that of the 3- and 4-methyl compounds. 2, 4-Dinitrophenoxyacetic acid also has some activity, whereas the mononitro derivatives have none. Trisubstituted compounds are in general even less active than comparable disubstituted derivatives. Introduction of additional ring substituents may lead to complete loss of activity in some, though not in all, cases. Propionic and butyric homologs are usually much less active than the related phenoxy- acetic acids although in some instances the reverse may be true. Replacement of the ether O atom by N greatly diminishes activity, while replacement by S nullifies it entirely. Presence of a free carboxyl group is not essential for formative activity. Certain of the amides and esters exhibit even greater activity than the parent acids. Relatively high activity may be shown by some acid chlorides. Halogen-substituted phenoxy ethanols, ethyl ethers, and ethylamines also may possess activity though of a relatively low magnitude. The phenoxy series provides the most comprehensive group enabling comparison of the structural requirements for stimulation of cell elongation and for formative effects. The curious situation exists that certain of these compounds have high activity in both types of response, whereas others possess auxin activity but not formative activity. Too few naphthoxy acids have been tested to provide detailed information on the influence on formative activity of small changes in the molecule. As already noted, ring substitution is not essential for activity in this series. No activity is shown by benzoic acid or any of its derivatives containing the following single substituents in the ring: amino, bromine, chlorine, fluorine, hydroxyl, iodine, nitro, carboxyl, and methyl. Some of the di- and tri- substituted derivatives are active however. In Table VI are listed active and inactive compounds of this type. A further group of diverse compounds without activity in this test is given in Table VII.

60 TABLE VI FORMATIVE ACTIVITY OF SOME DI- ANfl TRI- SUBSTITUTED BENZOIC ACIDS Active 2., 5-Dichlorobenzoic acid 5-Bromo-2-iodobenzoic acid 2-Bromo-3-nitrobenzoic acid 3 - Bromo-2-nitrobenzoic acid 3-Bromo-4-nitrobenzoic acid 2-Chloro-3-nitrobenzoic acid 2 -Iodo-3-nitrobenzoic acid 2-Methyl-3-nitrobenzoic acid 5-Bromo-3-chloro-2-iodobenzoic acid 5-Bromo-2, 3-dichlorobenzoic acid 5-Chloro-2, 3-dibromobenzoic acid 2, 3-Dibromo-5-iodobenzoic acid 3, 5-Dibromo-2-iodobenzoic acid 3, 5-Dichloro-2-iodobenzoic acid 2, 3, 5-Tribromobenzoic acid 2, 3, 5-Trichlorobenzoic acid 3, S-Dicarboxybenzoic acid 2, 4-Dichlorobenzoic acid 3,4-Dichlorobenzoic acid 2,4-Dihydroxybenzoic acid 2, 5-Dihydroxybenzoic acid 2, 5-Diiodobenzoic acid 3,4-Diiodobenzoic acid 2, 3-Dimethoxybenzoic acid 2f 5-Dinitrobenzoic acid 3, 5-Dinitrobenzoic acid 2-Amino-5-iodobenzoic acid 5 -Amino-2 -hydroxybenzoic acid 2-Bromo-4-nitrobenzoic acid 2-Bromo-5-nitrobenzoic acid 3-Bromo-5-nitrobenzoic acid 3 -Bromo-6 -nitrobenzoic acid 4-Bromo-2-nitrobenzoic acid 4-Bromo-3-nitrobenzoic acid 2-Fluoro-3-nitrobenzoic acid 2-Hydroxy-3-methyIbenzoic acid 2-Hydroxy-3-nitrobenzoic acid S-Bromo-Z-hydroxybenzoic acid 2-Hydroxy-S-iodobenzoic acid 5-Methyl-2-nitrobenzoic acid 2-Amino-3, 5-diiodobenzoic acid 2-Broxno-3, 5-dinitrobenzoic acid 2-Fluoro-3, 5-dinitrobenzoic acid 3-Bromo-2-hydroxy-5-sulfobenzoic acic 3, 5-Dinitro-2-hydroxybenzoic acid 2-Hydroxy-5-iodo-3-methylbenzoic acid 2,4, 6-Trihydroxybenzoic acid 2,4, 6-Trinitrobenzoic acid 3,4, 5-Tribromobenzoic acid 3,4, 5-Trichlorobenzoic acid 3,4, 5-Trihydroxybenzoic acid 3,4, 5-Triiodobenzoic acid

61 3, 5-Diiodo-2-hydroxybenzoic acid 3, 5-Diiodo-4-hydroxybenzoic acid TABLE VI (Cont. ) Inactive 3, 4. 5-Trimethoxybenzoic acid TABLE VII MISCELLANEOUS COMPOUNDS INACTIVE IN FORMATIVE RESPONSE Phenylacetic acid 4-Aminophenylacetic acid 4 - Bromopheny lacetu acid 4-Chlorophenylacetic acid 4-Hydroxypheny'acetic acid 4-lodophenylacetic acid 4-Nitrophenylacetic acid a-(2-Hydroxyphenyl)-phenylacetic acid 2, 4-Dinitrophenylacetic acid 4-(Trimethylsilyl)-phenylacetic acid Diphenylacetic acid p-Methallyldiphenyl acetic acid Allyldiphenylacetic acid Hydrocinnamic acid cis-Cinnamic acid trans -Cinnamic acid 2-Chlorocinnamic acid 4-Chlorocinnamic acid 2, 4-Dichlorocinnamic acid 2-Methoxycinnamic acid cis -5-Chloro-2 -hydroxycinnamic acid a-Phenylbutyric acid 4-Hydroxyphenylglycine 3, 5-Dibromo-L-tyrosine Coumarilic acid 2-Thiophenecarboxylic acid 3-Pyridinecarboxylic acid (Nicotinic acid) Ni c otinam ide 4-Pyridinecarboxylic acid (Isonicotinic acid) 2-Pyriolidinecarboxylic acid (Proline) 1 -Naphthoic acid 2-Naphthoic acid 1-Hydroxy-2-naphthoic acid 3-Hydroxy-2-naphthoic acid 1 -Naphthylacetic acid 2-Naphthylacetic acid a-(l-Naphthyl)-propionic acid Naphthalene-1, 8-dicarboxylic acid Indole-3-acetic acid

62 TABLE VII (Cont. ) Tryptophane 8-Quinolyloxyacetic acid 5, 7-Dibromo-8-quinolyloxyacetic acid 9-Allylfluorene-9-carboxylic acid The impression may be given that the structural requirements for compounds active in producing formative responses are rather clearer and more restricted than for auxin activity or root initiation, and while this may be the case, it cannot be said to be proved at this time. The characterization of formative responses is a matter of great difficulty. Gross morphological abnormalities are not readily expressible. Certain compounds not containing phenoxy, naphthoxy, or benzoic groupings do induce changes in growth habit, but have not been included in comparative tests, so that their relative position is not known. In this category are such herbicides as maleic hydrazide, dichloral urea, and trichloroacetic acid. VII. CONTROL OF ABSCISSION There is a substantial body of evidence indicating that the process of abscission - the natural separation of leaves, foliage branches, floral parts, or fruits from the plant brought about by structural alterations in particular groups of cells - is regulated by a hormonal mechanism. Application to the plant of certain growth-regulatory compounds (Table VIII) has the effect of greatly delaying the onset of abscission. Such treatments have found extensive use for prevention of preharvest drop of orchard fruits and related purposes. TABLE VIII COMPOUNDS EFFECTIVE IN RETARDING ABSCISSION Levulinic acid Indole-3 -acetic acid p-(Indole-3)-propionic acid v-(Indole-3)-.n-butyric acid (also esters) Naphthalene Naphthalene-1 -acetic aciti (also esters) Naphthalene-1 -acetamide Naphthalene -1 -thioacetamide 1,2, 3,4-Tetrahydronaphthalene-6-acetamide Naphthoxy-2-acetic acid f3-(naphthoxy-l)-propionic acid 4-Chlorophenoxyacetic acid 2, 4-Dichlorophenoxyacetic acid (also esters) o-(2, 4-Dichlorophenoxy)-ii-butyric acid 2-Methyl-4-chlorophenoxyacetic acid

63 On the other hand, other compounds are known to produce the opposite effect, namely, to accelerate or induce abscission. One of these, ethylene, is of particular interest in that it is a plant product and conceivably may play a role in the natural control of abscission. Additional compounds reported active in inducing abscission include calcium cyanamide, ammonium thio- cyanate, carbon tetrachloride, ethylene chlorohydrin, 3, 6-endoxotetrahydrophthalic acid, 3,6- endoxohexahydrophthalic acid, and various halogenated benzoic acids. The evidence available from several types of investigations appears to support the view that abscission of an organ is induced when its content of auxin falls to a low level. The process may be retarded by supplying externally a substance with auxin activity and may be accelerated by any of a variety of means, physical as well as chemical, which result in diminution of the normal auxin content. The diversity of the chemical agents which can act as abscissants suggests that reduction of the auxin level can be accomplished by various mechanisms. Only in the benzoic acids has the relation of activity to structure of a closely related series of compounds been investigated 36 No activity is shown by benzoic acid itself or by any of its monohalogenated derivatives. Moderate activity is possessed by certain of the dihalo- genated compounds while a number of the trihalogenated benzoic acids are highly effective (Fig. 1). It is worthy of comment that here the halogens arrange themselves in the order I > Br > Cl in activating influence, which is in the reverse direction to that found in the phenoxy- acetic series in formative activity. MOLAR ABSCiSSi0N-iNDUCiNG ACTiViTY 4.00O 6.0O0 B.O00 ta.°°° * I Cl B. B, , I Cl I Cl Br I Br Cl I Cl Cl I I a ci Br Br Cl Cl Br Cl Cl Bf Br Cl Cl » I Cl Bf Br Cl Cl Br Cl Br Cl Cl Cl Fig. 1 . Activity of 2, 3, 5-trihalogen benzoic acids in inducing abscission

64 VIII. CONTROL OF BUD DEVELOPMENT A situation somewhat analogous to abscission is to be found in the control of bud development, which is also of great importance in the ontogeny of the plant. Here also dormancy appears to be under hormonal regulation. Endogenous auxins and related exogenous substances have the ability of maintaining or inducing dormancy, i.e. of suppressing bud development, whereas the reverse process of bud stimulation, or release of dormancy, can be accomplished by a congeries of rather diverse chemicals. Among the compounds active in suppressing bud development are indole-3-acetic acid, p-(indole-3)-a-oximino-propionic acid, p-(indole-3)-acry lic acid, p-(indole-3)-pyruvic acid, y- (indole-3)-.a-butyric acid, naphthalene-1-acetic acid (and its esters), naphthalene -1 -acetamide, naphthalene -1 -thioacetamide, and coumaran-1 -acetic acid. Although the number of compounds tested for activity of this kind is quite small, there appear to be rather narrow structural requirements; for example, c_is -cinnamic acid which exhibits auxin activity in cell-elongation tests is inactive in suppressing bud development. A number of compounds active in overcoming bud dormancy are listed in Table IX. TABLE IX COMPOUNDS EFFECTIVE IN OVERCOMING DORMANCY OF BUDS Diethyl ether Chloroform Ethyl bromide Ethyl iodide Ethylene chloride Ethylene chlorhydrin Ammonia Ethylene Trichloroethylene o-Tolylthiourea Ammonium dithiocarbamate Thiosemicarbazide Hydrogen sulfide Ethyl mercaptan Thioglycol Sodium azido-dithiocarbonate Methyl disulfide Potassium sulfocarbonate Thioacetamide Ethyl carbylamine Thiourea Propylene Acetaldehyde Ethanol Acetone Hydrogen cyanide Zinc sulfate Thiocyanates

IX. REPRESSION OF ROOT ELONGATION AND OF SEED GERMINATION The repression of root growth of seedlings has been employed for many years as a technique for investigating inhibitory chemicals. More recently such methods have been employed extensively in preliminary screening for compounds with herbicidal activity. The root shows high sensitivity to a great diversity of substances, however, so that little purpose would be served by an enumeration of all the compounds found to exert repressive effects. Interest in this response stems in part from the fact that certain of the compounds possessing auxin activity, e.g. indole -3 -acetic acid and 2, 4-dichlorophenoxyacetic acid, are among the most active root inhibitors, producing measurable effects in concentrations as low as 10'° to lO'^M. Up to the present, however, the number of compounds that have been carefully assayed in both types of response is too few to warrant discussion of the comparative structure - activity relations. A quite different class of substances, the arylcarbamic esters, also possesses activity of the same order in repression of root elongation but appears to be without effect in the other types of growth-regulatory phenomena so far discussed The following generalizations concern- ing the carbamates are based upon preliminary tests at Camp Detrick. The very low solubility of many of these compounds presents a major methodological difficulty which may have introduced some bias into the data available. Hence, these conclusions are to be regarded as provisional. Esters of carbamic acid itself are relatively inactive. Activity is conferred by substitution of one of the amino hydrogens by a phenyl radical or, in even greater degree, by certain substituted phenyl groups, particularly those containing halogen atoms. Compounds in which the second amino hydrogen is substituted, as by a methyl group, also may exhibit activity. The nature of the esterifying radical plays a role which appears to depend upon the type of N substitution. Thus, among the N-phenyl carbamates the isopropyl ester is most active, followed closely by ethyl and various p-substituted ethyl esters, whereas in other series the n.-propyl ester may equal or exceed the isopropyl in activity. Several thionocarbamates are active while the corresponding thiolcarbamates are not. N, N-di-(carboethoxy)-aniline, which contains a second esterified carboxyl group, has been reported of equal activity with the related ethyl N-phenylcarbamateZ . Closely related to suppression of root elongation is the inhibition of seed germination. The most obvious manifestation of germination is the elongation of the axes - root and shoot - of the embryo and this is commonly utilized as the criterion of the occurrence of germination. Ordinarily, root emergence precedes that of the shoot. At first, elongation of the root is due entirely to enlargement of cells pre-existing in the embryo but this initial phase merges so smoothly into that characteristic of subsequent growth, in which formation of new cells proceeds concomitantly with their expansion, that macroscopic observation permits no distinction between them. Hence separation of the germination stage from that of seedling development is entirely arbitrary. Whether there exist compounds capable of controlling specifically the initial phase of germination but inactive on later root growth is not clear at present. Substances highly inhibitory to seed germination have been shown to occur in fruits and other organs of a wide variety of plants . There is every reason to believe that some of these play an important role in the control of development. The term "blastokolin", which was introduced originally 5 to designate the germination-inhibiting substance present in the flesh of certain fruits, has been employed by some later workers in a generic sense. While the nature of many of these substances is still unknown, a number have been identified. Several classes of organic compounds are represented, including mustard oils, cyanophoric glycosides, aldehydes, carboxylic acids, essential oils, alkaloids, phenols, and unsaturated lactones. Relatively high concentrations, of the order of 10*2 to 10-3M, of most of these substances are usually required for inhibition of germination The wide diversity of structural types which exhibit activity of this kind bespeaks a multiplicity of inhibitory mechanisms having a common result.

Only one of these groups of compounds - the unsaturated lactones - will be further considered. The most active of these are effective at concentrations of 10*4 to 10*5 M in root repression and at somewhat higher concentrations in prevention of germination. In Table X arc shown a number of lactones which possess blastokolin activity. As these have been assembled from various investigations', 6, 11, 18, 22, 34 no comprehensive comparison of activities is possible. However, so far as can be judged, the most active of these compounds is protoanernon- in, followed by coumarin. Substitution in either ring of the latter compound is stated to diminish activity. The diversity of structural types suggests that activity is a general property of a, p- unsaturated lactones; the relative activity is of course influenced by the specific structure of the whole molecule. TABLE X UNSATURATED LACTONES EXHIBITING BLASTOKOLIN ACTIVITY H 'C*+ H2C ^CH CH3HCX s-C = O Hexene-2-olid-5, 1 (parasorbic acid) HC—CH I 1 C = O V Pentene-2-olid-4, 1 H2 S-C^ HC CH2 sO" Hexene-4-ohd-5, 1 HC=— -CH HC CH X C=O P r otoane monin HC CH CH3CH2CH2HCX C = O Heptene-2-olid-4, 1 Hexene-2-olid-4, 1 H C CH HC — CH =o 4-Methylpentene-2-olid-4, 1 HC-=CH Pentene-2-olid-5,l ( 3 -Pentenolactone) CH2-CH2 Lactone of 2-(l' -Hydroxycyclohexyl)acrylic acid

67 CH ,C=O TABLE X (Cont. ) H He' 3-Hydroxy-a-pyrone H TH Daphnetin V CH3 CH3C 4, 6-Dimethyl-a-py rone H *C-COOH Coumarin-3-carboxylic acid H 'C* 6-Phenyl-a-pyrone OH HC^ VC-COCH3 Dehydroacetic acid OH =0 3-Acetyl-4-hydroxycoumarin H Cl 'CH 6-Chlorocoumarin 8" Dicoumarol Coumarin Esculetin

68 The root-inhibiting activity of an extensive series of coumarin derivatives has recently been studied^. Coumarin-3-carboxylic acid, 8-methylcoumarin, 7, 8-dihydroxycoumarin (daphnetin), and 7, 8-dihydroxy-4-methylcoumarin were found fully as active as coumarin itself. All the other compounds tested, nineteen in number, were appreciably less active. These included all the monomethyl isomers and various hydroxy, methylhydroxy, and methoxy sub- stituted coumarins. X. GENERAL DISCUSSION Interest in the relationship between structure and activity stems from two principal sources. The increasingly widespread utilization of plant growth-regulators for practical ends in recent years has intensified the interest in this subject with the hope that promising directions of search for additional useful compounds may be indicated. In addition, it is expected that insight into the structural requisites will contribute to understanding of the mechanisms concerned in the regulation of plant development. To date neither of these hopes has been fulfilled. The search for new types of useful growth-regulators, which is being vigorously prosecuted in many laboratories, appears to be largely by trial and error. The complexity of the physiological systems involved in the various processes influenced by plant growth-regulators has thus far defied interpretation on a biochemical level; in conse- quence there have been proposed a number of more or less incompatible hypotheses some of which appear to be based upon insecure experimental foundation. While an extended considera- tion of hypothetical mechanisms would be out of place in this paper one or two main points should be mentioned. Arising primarily from work on plant tropisms and correlation, there has developed in the past quarter-century the concept of a system of endogenous growth-regulators in plants which control in a hormone-like manner many of the features of growth and development. It is believed that substances of high potency may be formed in certain organs, such as the bud or the leaf, and regulate or control normal phenomena of difterentiation and development as well as many of the growth adjustments which follow injury or organ removal. The nature and mode of action of such growth-regulators is very imperfectly understood. Omitting the case of ethylene, to which reference has already been made, only one substance, indole -3-acetic acid, or heteroauxin, has been certainly identified as an endogenous phytohormone. The versatility of this compound, an indication of which has been given in the foregoing sections, has led some investigators to the view that it may play a central and direct role in all, or most, manifestations of growth and development. Others believe that there exist in the plant diverse growth regulators, more or less specific for particular developmental phenomena, the function- ing of which may be influenced by heteroauxin; thus a more indirect role is ascribed to the latter. Opinions as to the fundamental mode of action of this phytohormone may be divided into two general categories. On the one hand it is suggested that heteroauxin participates as a co- enzyme in some enzymatic metabolic reaction, a so-called master reaction, common to various kinds of plant cells and leading to diverse end-results according to environmental conditions and the physiological and morphological potentialities of the particular cells involved. It cannot be said that this view, which may be termed for convenience the biochemical hypothesis, is supported by any convincing evidence of direct nature. On the contrary, analogy with known cases of coenzyme action seems to speak against it. The other suggestion is that the primary action of heteroauxin is to affect some more physico-chemical property of the cell, such as the degree of association or dissociation of protoplasmic proteins. In this way exposure or masking of active sites on enzymes might be brought about with resulting increase or decrease of enzyme activity. Further, the concomitant changes in structural viscosity of the protoplasm might be expected to influence physiological

69 processes such as cell expansion, permeability, and protoplasmic streaming. The postulated physico-chemical mechanism may be thought of as in some measure nonspecific with regard both to the active agents and to the types of proteins or enzymes affected. This view provides an explanation of the manner in which teratological and lethal effects can be produced by high dosages of compounds which at lower rates may act similarly to endogenous hormones in normal growth processes. A physico-chemical mode of action .tf juld appear to harmonize with the essentially physical requirements for auxin activity as postulated by Veldstra. Whether the mode of action of growth-regulators be primarily biochemical or primarily physico-chemical, it must involve reactions peculiar to the green plant. Although comparative biochemistry does reveal similarities in the biocatalysts and the enzymically catalysed reactions involved in oxidations and energy transfer in unrelated groups of organisms, it must be pointed out that the degree of specificity of growth-regulators is apparently high. Few are known to influence organisms other than higher plants at comparable dosages though it must be admitted that the data on this point are extremely scanty. The mechanism of action of exogenous growth-regulators will probably not be fully explicable until that of the endogenous growth hormones is understood. Nevertheless, knowledge of the structural requisites of the former may be very helpful in elucidating the role of the latter. There can be little doubt that in many cases the exogenous compounds act by participation in the same mechanism by which the native hormone operates. Thus, in some responses, such as stimulation of cell elongation, initiation of roots, induction of parthenocarpy, inhibition of bud development, and retardation of abscission, the exogenous growth-regulators appear capable of functioning as substitutes for the endogenous hormone. In certain other responses the relation- ship is less clear. In responses such as modification of leaf form, induction of abscission, and stimulation of bud development, the effects of the exogenous growth-regulators are the converse of those brought about by indole-3-acetic acid; in these phenomena, if the two classes of substances do act through a common basic mechanism, the relationship may be one of antagonism. There is ; ome evidence that such is the case.

70 LITERATURE CITED No attempt has been made to document completely all the results included in this paper Citations have in general been restricted to the more recent publications and to other summariz- ing articles in which references to most of the original literature may be found. 1. Audus. L.J. and Quastel, J. H. , Nature, 159. 32O(I947). 2. Bentley, J. A. , Nature, 165. 449(1950). 3. Bonner, D. M. , Botan. Gaz., 100. 200(1938). 4. Booij, H.L. and Veldstra, H. , Biochim. Biophys. Acta. 3. 260(1949). 5. Brown. J.W. and Weintraub, R. L. , Botan. Gaz., 111. 448(1950). 6. Cameron, F. K. , J. Phys. Chem. , 14, 422(1910). 7. Evenari. M. , Botan. Rev., 1J, 153(1949). 8. Findlay, S. and Dougherty, G. , J. Biol. Chem., 183. 361 (1950). 9. Goodwin, R.H. and Taves, C. . Am. J. Botany, 37, 224(1950). 10. Haagen Smit, A.J. and Went, F. W. , Proc. Acad. Sci. Amsterdam, 38, 852(1935). 11. Haynes, L.J. and Jones. E.R.H. , J. Chem. Soc. . p. 954(1946). 12. Irvine, V.C., Univ. Colorado Studies, 26., 69(1938). 13. Ivens, G. W. and Blackman, G. E. , Symp. Soc. Exp. Biol., 3, 266(1949). 14. Jones, R. L. , Metcalf, T.P. and Sexton, W.A. , Biochem. J. , 45, 143(1949). 15. Kockemann, A., Ber. deut. bot. Ges. , 52, 523 (1934). 16. Koepfli, J.B., Thimann, K. V. and Went, F. W. , J. Biol. Chem., 122. 763(1938). 17. Kbgl, F. and Kostermans, D. G. F. R. , Z. physiol. Chem., 235. 201 (1935). 18. Kuhn, R. et al, Naturwissenschaften, 31, 468 (1943). 19. Luckwill, L. C., J. Hort. Sci., 24, 19(1948). 20. Muir, R.M., Hansch. C. H. and Gallup, A. H. , Plant Physiol. , 24, 359(1949). 21. Sexton, W.A., Chemical constitution and biological activity, London (1949). 22. Sigmund, W., Biochem. Z. . 62, 339(1914). 23. Skoog, F. , J. Gen. Physiol., 20, 311 (1937). 24. Snow, R., Nature, 139. 27 (1937). 25. Synerholm, M. E. and Zimmerman, P. W. , Contrib. Boyce Thompson Inst. , 14, 39, 91 (1945).

71 26. Synerholm, M. E. and Zimmerman, P. W. , Contrib. Boyce Thompson Inst. , 14, 369(1947). 27. Templeman, W.G. and Sexton. W. A. , Proc. Roy. Soc. (London). B, 133, 480(1946). 28. Thimann, K. V. , Plant growth substances (F. Skoog, ed. ), Univ. Wisconsin Press, 1951. 29. Veldstra. H. . Enzymologia, 1_1, 97(1944). 30. Veldstra. H. , Biochim. Biophys. Acta. 1. 364(1947). 31. Veldstra. H. , Bull. soc. chim. biol. , 31. 594(1949). 32. Veldstra, H. , and Booij, H. L. , Biochim. Biophys. Acta, .3, 278 (1949). 33. Veldstra, H. and Havinga. E. , Rec. trav. chim., 62, 841 (1943). 34. Veldstra, H. and Havinga, E. , Enzymologia, 11, 373(1945). 35. Watson, D. P. , Am. J. Botany, 35, 543(1948). 36. Weintraub, R.L. , Brown, J. W. , Nickerson, J. C. and Taylor, K. N. , 24th Annual Meeting, American Society of Plant Physiologists, (Dec. 29. 1949). 37. Went, F. W., Proc. Acad. Sci. Amsterdam, 42, 581 (1939). 38. Went. F. W. , Arch. Biochem. , 20, 131 (1949). 39. Went. F. W. , and Thimann, K. V. , Phytohormones, New York (1937). 40. Zimmerman. P. W. , Proc. Nat. Acad. Sci. U.S., 27, 381 (1941). 41. Zimmerman, P.W., Cold Spring Harbor Symposia Quant. Biol., 10, 152(1942). 42. Zimmerman, P. W. , Ind. Eng. Chem. , Ind. Ed., 35, 596(1943). 43. Zimmerman, P. W. , Torreya, 43, 98(1943). 44. Zimmerman, P. W. , in Growth of plants (W. Crocker, ed. ), New York (1948). 45. Zimmerman, P. W. and Hitchcock, A. E. , Contrib. Boyce Thompson Inst. , 1_2, 1 (1941). 46. Zimmerman, P.W. and Hitchcock, A. E. , Contrib. Boyce Thompson Inst. , 1_2, 321, 491 (1942). 47. Zimmerman, P.W. and Hitchcock, A. E. , Contrib. Boyce Thompson Inst. , 1_3. 313(1944). 48. Zimmerman, P.W. and Hitchcock, A. E. , Contrib. Boyce Thompson Inst. , 15, 421 (1949). 49. Zimmerman, P.W., Hitchcock, A. E. and Harvill, E. K. , Contrib. Boyce Thompson Inst. , 13, 273 (1944). 50. Zimmerman, P. W. and Wilcoxon, F. , Contrib. Boyce Thompson Inst. , J, 209 (1935).

72 DISCUSSION DR. D. W. WOOLLEY (Rockefeller Institute for Medical Research, New York. New York): Mr. Chairman, in thinking of the relationship of structure to activity and, more particularly, in attempting to make generalizations about the observed data, I wonder if we can be too narrow in our view of the matter. I thought of this particularly when the activity of the naphthoic arid was mentioned. In looking at a naphthalene ring, 'I wonder if we cannot regard it in two fashions: one, as a benzene ring, doubly substituted with an unsaturated side chain. In some respects it is analogous to dimethyl benzene. On the other hand, in looking at a naphthalene ring, I wonder if we cannot regard it as a benzene ring with a long alkyl side chain branch, part of it bent around and fused onto the benzene ring. These may be mental gymnastics but, occasionally, it seems to me, they help in trying to formulate in our minds at least some order in the vast array of compounds which show a given activity. DR. McKEEN CATTELL^Cornell University Medical College. New York, New York): I would like to comment on one point which seems to me of some importance to our general problem, and that is the question of the emphasis that should be given to the exceptions in the structural requirements necessary for a given activity. These exceptions are always challenging, but it seems to me that, when we consider the complexity of the biological systems concerned, it need not be disturbing to find outstanding exceptions in structural relationships. If we list the numerous factors which may influence the particular biological properties under observation, such as in the study of effects of growth, it becomes apparent that similar effects might be produced by many different types of action. It is evident that such exceptions are of interest not only in relation to further chemical development but particularly in relation to the analysis of the mechanism and site of action of an agent. Of course, to a certain extent, these complexities are eliminated when we are dealing with the simpler enzyme systems, and in these cases the correlation between structure and activity may be more significant.

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First Symposium on Chemical-Biological Correlation is a summary of a symposium held on May 26-27, 1950 by the Chemical-Biological Coordination Center. The purpose of the symposium was to bring together scientists trained in chemistry and biology for discussion of problems concerned with the effect of structure of chemicals on their biological activity and the mechanism of such actions.

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