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II.I.I. {(6.460 hilt- t. .4 .. . ..n.r.r.:f...l>\z VA- , .. MilHlllllllfllfllllllfllflljfllIllillllfllflll Inll’l!!!)K‘f Michiyn State University 1‘ This is to certify that the thesis entitled THE PHYSIOLOGY AND ANALYSIS OF INDOLE-3-ACETIC ACID AND ITS MYO-INOSITOL ESTERS presented by Jerry David Cohen has been accepted towards fulfillment of the requirements for Ph.D. dpgvpin Botany and PTant Pathology 7 o QK'MA‘A g- JDAA dam/”e Major professor Date w 0-7639 THE PHYSIOLOGY AND ANALYSIS OF INDOLE—3-ACETIC ACID AND ITS MYO-INOSITOL ESTERS By Jerry David Cohen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1979 //(,_i',O ABSTRACT THE PHYSIOLOGY AND ANALYSIS OF INDOLE-3-ACETIC ACID AND ITS MYO-INOSITOL ESTERS By Jerry David Cohen 1. The Bound Auxins: Protection of IndOZe-S-acetie Acid from Peroxidase—catalyzed OxidatiOn. Indole-3-acetic acid (IAA) was oxidized by horseradish peroxidase, but ester and amide conjugates of IAA were not degraded. Addition of indoleacetyl-mya-inositol, indoleacetyl-L—aspartate, indoleacetylglycine, indoleacetyl-L- alanine, indoleacetyl-D-alanine, or indoleacetyl-B-alanine did not affect the rate of oxidation of IAA by horseradish peroxidase. Peroxidase preparations from Pisum sativum L. and Zea mays L. behaved similarly in that they rapidly oxidized IAA, but not con- jugates found in the plant from which the peroxidase was prepared. These results indicate that conjugation could affect the stability of IAA in vivo. II. Photo—regulation of the Ratio of Ester to Free Indole- 5-acetie Acid. A light exposure, sufficient to cause a 30% reduc- tion in growth rate of seedlings of Zea mays, causes a decrease of 40% in the concentration of free indole—3—acetic acid in the seed— ling and an increase in the content of esterified indole—3—acetic Jerry David Cohen acid. It is concluded that one mechanism for regulation of plant growth is alteration of the ratio of free to conjugated indole-3- acetic acid by environmental stimuli. III. Synthesis of 14C-IndoZe-3-acetyZ—mugfinositoZ. 14C-indoleacetyl-myo-inositols Synthesis of the mixed isomeric from carrier-free B[2-14C]-indoleacetic acid (57.2 mCi/mmole) and inositol via an imidazolide intermediate is described. Radiologi— cal decomposition of the indolylic compounds was prevented by the use of a volatile thiol, dithioethane, and anthracene. IV. Automated Analysis of IndoZyZic Compounds in Plant Extracts. An automated procedure for analysis of indolylic com- pounds has been developed utilizing a chromogenic reagent yielding stable chromogens with Egm = 24,000. Coupling this detection pro— cedure to an adsorption chromatographic system of sulfonated poly- styrene divinylbenzene, such as used in automated amino acid analysis, permitted separation, detection and assay of indoles in crude extracts of kernels of Zea mays. Analysis of the major neutral and acidic indoles in crude extracts was accomplished in less than 3 hours, where previous methods required several days. Adaptability of this method to other tissues and column chromato- graphic systems is described. These methods describe the first versatile indole analyzer useable with crude plant extracts. The analyzer should have significant applications in assays of indoles in both plant and animal extracts. Jerry David Cohen V. Double Standard Jeotope Dilution Assay I. Quantitative Assay of Indole-3-acetic Acid. Isotope dilution analysis for the quantitation of labile compounds has been limited by the amount of sample necessary to redetermine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determination of specific activity by gas chromotography. It requires the availability of the radiolabeled compound to be analyzed and also requires a chemically—related radiolabeled com- pound. The method is illustrated by assaying indole-3-acetic acid 14C— in plant extracts using 14C-indole-3—acetic acid and adding indole-3—butyric acid at the final stage of analysis prior to gas chromatography. Used with a nitrogen specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of 14C-[2-ring]-indole-3-butyric acid is also des— cribed. To my parents, my brother, my sister, and my wife; together we share troubled and joyful times. ACKNOWLEDGMENTS For his support, enthusiasm and friendship I will always fondly remember my major professor, Dr. Robert S. Bandurski. His constant drive to understand biochemical systems should serve as an inspiration to all scientists. Dr. Norman E. Good has, in addition to serving on my committee, provided helpful discussions, showed concern and offered advice for which I am indebted to him. The other members of my guidance committee, Drs. Robert P. Scheffer and Philip Filner, provided patient and scholarly advice for which I remain most grateful. Dr. Axel Ehmann provided an early auto— catalytic introduction to indole chemistry which led to many of these investigations. I thank Drs. Chapman, Sweeley, and Holland of the MSU/NIH mass spectrometry facility for their help and cooperation through- out this work. Also, Drs. Matthew Zabik, Richard Leavitt and Derek Lamport provided excellent advice on analytical techniques and Dr. Jane Shen-Miller of the National Science Foundation has been a constant source of information and assistance on the physiology of tropic behavior. Discussions with Axel, Greg, Peter and Frank were among the most memorable experiences of my early graduate training at Michigan State and working with Janusz, Lech, Prudy, Ephraim, and iii Volker was both a pleasure and an honor. Aga Schulze, a true pioneer in the quantitative analysis of indole-3-acetic acid in plants, has served as my teacher, colleague and friend during this thesis work. I have also benefited greatly from knowing many grad— uate students, postdocs and faculty at M.S.U. and only regret that space does not permit listing them all by name. I thank Bill Richards, Gail Ryder and all of the delightful people at the stores order desk for decreasing the time and effort required to complete this research. Lastly, I wish to express my love and thanks to my wife I Susan who has worked with me to complete this dissertation. TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES INTRODUCTION LITERATURE REVIEW . I. Oxidation of Indole—B-acetic Acid II. Environmental Effects on Indole-3-acetic Acid Levels III. Covalently Bonded Hormones . IV. Analytical Chemistry of Indole- 3- acetic Acid and Its Adducts . . . . . . EXPERIMENTAL I. The Bound Auxins: Protection of Indole-3-acetic Acid from Peroxidase-catalyzed Oxidation II. Photo-regulation of the Ratio of Ester to Free Indole-3-acetic Acid . . . . . . . . l4 III. Synthesis of C—Indole-3-acetyl-myo-inositol IV. Automated Analysis of Indolylic Compounds in Plant Extracts . . . . . . . . . V. Double Standard Isotope Dilution Assay 1. Quantitative Assay of Indole-B-acetic Acid CONCLUSIONS . BIBLIOGRAPHY Page vi vii TO 24 49 71 7l 77 82 87 ll4 I41 I47 LIST OF TABLES LITERATURE REVIEW l. Indole— 3- acetic acid and its adducts in Zea mays kernels . . . . . . . . . . . . 2. Some common paper chromatographic systems used for the separation of indole-3-acetic acid . 3. Thin layer chromatographic systems which have been used for the separation of indole-3-acetic acid 4. Gas—liquid chromatographic methods used for the separation and analysis of indole-3-acetic acid EXPERIMENTAL I l. The oxidation by horseradish peroxidase of IAA and IAA conjugates incubated for l h at 25° C . 2. Failure of IAA conjugates to inhibit the rate of free IAA oxidation by horseradish peroxidase (as measured by the change in A251) . . . 3. The oxidation of IAA and IAA conjugates by enzyme preparations from Zea and Pisum . . . EXPERIMENTAL II I. Photoinhibition of growth . II. Photo-induced change in free and free plus ester IAA . . . . . . . . . . . . . . . EXPERIMENTAL V l. Experimental values obtained with repetitive injec- tions using derivatization with BSTFA + % TMCS and flame ionization detection . . . . . . vi Page 26 53 56 62 73 74 74 78 79 I40 LIST OF FIGURES LITERATURE REVIEW l. Diagram of photoinhibition of growth which accounts for discrepancies found in the literature . EXPERIMENTAL I l. 2. Change in absorption spectra of standard reaction mixtures (see text) containing peroxidase and either IAA (A) or indoleacetyl—myo-inositol (B) A scheme for l) the destruction of excess IAA, and 2) the destruction of IAA once it has performed its growth promoting function (see discussion in text). EXPERIMENTAL II l. A diagramatic interpretation of the experiments of Elliot and Shen-Miller (22) and Shen-Miller et al., (27) indicating the difficulty in explaining photo- tropism as a consequence of lateral transport of IAA if phototropism and photoinhibition of growth are mechanistically related phenomenon EXPERIMENTAL IV l. Diagramatic representation of the PA-28 chromato- graphic system . . . . . . . . . Automated analyzer for the determination of indoles with the Ehmann reagent . . . . . . . The effect of the rate of solvent flow on the elution profile of a mixture of IAA and mixed isomeric IAA-myo-inositols from the PA-28 column as measured by U.V. absorbance . . . . The effect of column temperature on the elution profile of a mixture of IAA and mixed isomeric IAA—myo-inositols from the PA-28 column vii Page 73 75 80 l06 lO7 108 l09 EXPERIMENTAL IV, cont. 5. Absorbancy of the Ehmann product of IAA as a func- tion of wavelength using a Cary l5 spectro— photometer . . . Calibration of the autoanalyzer by injection into the sample stream and using 6.6 mm flow cells Thin layer chromatogram of fractions from the PA-28 column of a crude extract of Zea mays kernels Recording of the elution profile as visualized with the analyzer for a crude extract from Zea mays kernels . . . . . . . . EXPERIMENTAL V l. Reaction scheme for the synthesis of 4C- [2- ring]- indole- 3- butyric acid from 14C- [2— ring]— indole and y-butyrolactone in strong base Ultraviolet absorption spectrum for the synthetic 14C- [2— ring]— indole- 3- butyric acid after Sephadex LH- 20 chromatography . . 70 eV electron impact mass spectrum of the synthetic 14C- [2— ring]- indole- 3- butyric acid methyl ester . Nitrogen specific themionic detector recorder trac- ing of a mixture of IAA and IBA as the methyl esters . Methylated sample from corn seedlings analyzed with the flame ionization detector (F10) and the nitro- gen specific thermionic detector (N2) . . viii Page llO ll2 ll3 l35 l36 l37 l38 l39 INTRODUCTION Indole-3-acetic acid (IAA) is the major plant growth hormone "auxin" (K691 and Haagen-Smit, l93l)) found in higher plants (cf. review by Gordon, l954). Studies on auxins have been published for well over lOO years--predating even the first use of the word "hormone" (Starling, l905) by 30 years-—and yet its biosynthesis, degradation, and mode of action are still topics of research and debate. Indeed, the unequivocal demonstration of the presence of IAA in plant tissue occurred only in this decade (cf. Ueda et al., l970; Greenwood et al., l972) even though the total number of scientific papers on auxins probably now numbers nearly l0,000. The bulk of the IAA in all plants appears to be linked by either an ester or amide linkage to other compounds (Bandurski and Schulze, l977b), although few of these compounds have been identi— fied. At the time this study was initiated (l974/l975) little was known about the function of these conjugates of IAA, although earlier workers in this laboratory had identified all of the com- pounds in kernels of Zea mays down to a level of l0 ug/kg dry wt. (cf. review by Ehmann, l973). This study was initiated, therefore, with the objective of elucidating the function of the covalently linked conjugates of IAA in the normal hormonal physiology of corn seedlings. During the course of this work it became apparent that continued progress in understanding hormonal biochemistry required faster and more sensitive methods for analysis with the requisite selectivity. Development of some of these methods, therefore, forms a major part of this research. The experimental work is in the form of five individual research papers, three of which have been pub- lished, while the other two are in manuscript form.1 1The second and third reprints are included only for the convenience of the reader. Parts I, IV, and V constitute the 'corpus' of the dissertation. As Albert Einstein has said in dis- cussing university administrators (Anon., l979), "Let elephants live." LITERATURE REVIEW 1. Oxidation of Indole-3—acetic Acid For a hormone to regulate growth it must be limiting and the maximum rate of growth is determined by the supply of the hor- mone. Thus, it is necessary for the plant to destroy IAA once it has performed its growth promoting act. These relationships have been anticipated previously (Bonner and Thimann, l935; Galston and Hillman, l96l) and have formed the theoretical basis for a wealth of studies on the peroxidative oxidation of IAA (cf. reviews by Galston and Hillman, l96l; Hare, l964). Thimann (1934) provided the first evidence that plant tis— sues can inactivate auxin. He incubated Vicia faba and Helianthus leaf extracts in auxin solutions and showed decreased levels of auxins, while leaves of Malva, having lower levels of polyphenol oxidase, were less active in auxin destruction. Van Overbeek (l935) correlated auxin levels with the rate of which corn tissues degraded auxin. These early experiments led to a number of correlative studies which now predominate in the literature on auxin destruction (see review by Hare, l964). The first careful study of the nature of the auxin destroy- ing enzyme in plant extracts was that of Tang and Bonner (l947), although Larsen (l936, I940) earlier showed that the "auxin inacti- vating substance" was enzymatic and required oxygen for activity. 3 Tang and Bonner further characterized this activity and showed that the enzyme was an iron metalloprotein which oxidized IAA in a manner which retained the indole nucleus. They also established the spe- cific nature of the side chain involvement by showing that indole— acetamide, indolepropionic acid, indolebutyric acid and tryptophan were not oxidized and showed that other auxins did not inhibit IAA oxidation. Galston et al. (1953) extended these studies to show that the enzyme from peas which Tang and Bonner described was in fact a peroxidase (donor: H202 oxidoreductase, EC l.ll.l.7) and that crystalline horseradish root peroxidase also catalyzed the oxidation of IAA in the presence of H202. The enzymatic degradation of IAA by horseradish as well as other plant peroxidases has subsequently received considerable attention (see review by Ray, 1958) and a proposed pathway has been suggested by Hinman and Lang (1965) based on spectrophotometric and chemical evidence. The oxidation of IAA is concentration dependent (Hinman and Lang, 1965) and below 2 x 10-4 M the reaction yields predominately 3-methylene oxindole, whereas at higher concentrations the principal product is a neutral indole which has the properties of an ester between IAA and indole—3-carbinol. The formation of the 3-methylene oxindole is probably accomplished (Hinman and Lang, l965; Yamazaki et al., 1977) by peroxidase acting as a one-electron oxidizing agent and forming indolenine hydroperoxide as the first intermediate. This is then converted to an indolenine epoxide which goes on to oxindole-3—carbinol. The carbinol then undergoes a slow conversion to 3-methylene oxindole. BeMiller and Colilla (l972) proposed another mechanism in which the enzyme catalyzes a two elec— tron oxidation and results in the formation of a cyclic 5—membered peroxide at ring carbons 2 and 3. The peroxide rearranges to the B-keto alcohol which converts to 3-methy1ene oxindole spontaneously. BeMiller and Colilla also propose a skatyl peroxide as an inter- mediate in the formation of the minor product, indole-3-aldehyde, while according to Hinmann and Lang (1965) this is formed by the addition of a proton to the epoxide. The Hinman and Lang (1965) proposal is more consistent with known indole chemistry Since oxi- dation at the 2-position usually results in ring-opening and because attack at the electron dense 3-position should be favored. Both proposals involve as a first step a decarboxylation which would be inhibited by esterification or amide linkage at the carboxylic acid (Hamilton et al., 1976; Cohen and Bandurski, 1978). Hinman and Lang (1965) report IAA-ethyl ester oxidation at a very slow rate, but this could be due to an internal ester hydrolysis. By whatever route IAA is oxidized by peroxidase it represents a rare example of indole oxidation not involving cleavage of the hetero ring. In the few studies in which the contribution of the decarboxylation route to IAA destruction has been studied in vivo it appears to be a minor route, accounting for only 15—30% of the IAA degradation (Davies, 1973; Epstein and Bandurski, 1978). The idea that peroxidase oxidation of IAA may simply be an artifact of cutting or homogenization (Briggs et al., 1955; Bonner, 1957) certainly cannot be excluded. The peroxidative route, however, probably becomes more important during injury (Haard and Marshall, 1976), tissue aging (Galston and Dalberg, 1954; Pilet and Galston, 1955), in tissue culture (Epstein et al., 1975) at cut surfaces and during tissue homogenization (Went, 1928), and when tissue is sub- jected to large amounts of exogenous IAA (Galston and Dalberg, 1954). It would appear that a peroxidative decarboxylation is the major degradative pathway for IAA provided to tissue cultures since Epstein et al. (1975) showed 90% decarboxylation in 4 hrs with 3 month old apple callus. Hamilton et al. (1976) demonstrated that 53% of the IAA supplied to cultures of Parthenocissus was decarboxy— lated within 48 hrs and they showed that labeled products of peroxi- dase degradation of IAA were found in the tissue and media. Sub- strates such as indole—3-propionic acid, indole-3-buytric acid, IAA—ethyl ester, tryptamine, and tryptophan are usually found to be relatively immune to peroxidative attack (Tang and Bonner, 1947; Hinman and Lang, 1965) and the prolonged efficacy of some synthetic auxins, as well as naturally occurring 4-chloro-IAA, has been attributed to this resistance (Marumo et al., 1974; Thimann, 1977). This may well be true in some systems like tissue culture and root— ing, but the evidence in many studies is ambiguous since IAA is also more easily oxidized by chelate formation with metals (Hinman and Lang, 1965). Whether peroxidase and ”IAA oxidase” activity always reside on the same enzyme is still unresolved, although recent work with high resolution isoelectric focusing and electrophoresis has begun to answer this question. Hoyle (1977) has shown that commercial horseradish peroxidase preparations contain 42 isoenzymes, all with both activities. It would appear, therefore, that all horseradish peroxidases are capable of IAA oxidation, although several earlier reports of plant peroxidase without IAA oxidizing activity have appeared (Endo, 1968; Yoneda and Endo, 1969; Sahulka, 1970; Frenkel, 1972). Are there enzymes, however, which oxidize IAA by a similar manner as peroxidase but which lack peroxidase activity? This is difficult to evaluate since much of the work on IAA oxidizing systems utilize methods which simply measure the loss of IAA without regard for the products produced. Several papers, however, have reported separation of IAA degrading activities which do not have peroxidative activity when tested with one or more model redox dye systems (Sequeira and Mineo, 1966; Van der Mast, 1969; Bryant and Lane, 1979). These observations may have their explanation in the differential sensitivity of the assays employed. Aside from peroxidase catalyzed oxidation other possible degradative routes for IAA have not been closely examined. How— ever, on purely chemical grounds (Remers, 1972; Smith, 1972), ring opening between C(2) and C(3) might be a likely route. Nair and Vaidyanathan (1964) have described an indole 2,3-dioxygenase (indole:oxygen 2,3—oxidoreductase E.C. 1.13.11.17) from leaves of Tecoma stans which adds 02 across C(2) and C(3) of indole to give N—formyl—o-aminobenzaldehyde. This same enzymatic activity is also present in leaves of Zea mays (Chauhan et al., 1978) and is analogous to the reaction of tryptophan 2,3 dioxygenase (L-tryptophanzoxygen 2,3 oxidoreductase E.C. 1.13.11.11) which forms L—formylkynurenine from L-tryptophan (Tanaka and Knox, 1959) and it is also analogous to the indoleamine 2,3 dioxygenase des- cribed by Hirata et al. (1977). Indole oxidizing enzymes of Tecoma and Zea are cuproflavoproteins whereas the tryptophan and indole— amine 2,3 dioxygenases are heme metalloproteins. Both the Tecoma and Zea enzymes appear to give anthranil (2,1-benzisooxazole) as the final product, suggesting the formation of o-aminobenzaldehyde from N-formyl—o-aminobenzaldehyde and the further oxidation to anthranil. Anthranilic acid was also identified as a product of the Zea enzyme although it apparently is not formed by the enzyme from Tecoma. If IAA were metabolized in an analogous reaction to that catalyzed by indole-2,3-dioxygenase then one might expect a product such as N-formyl-o-aminobenzoylacetic acid: 0 C-CHZ-COOH NH-CHO This could be metabolized to provide entry into the C6-C3 phenyl- propanoid pathways (cf. Geissman and Crout, 1969). A less likely route for IAA degradation would be removal of the side chain in a reaction similar to that catalyzed by tryptophanase (Happold, 1950) (L-tryptophan indole-lyase E.C. 4.1.99.1) to yield free indole which could proceed as recounted above. Kinashi et al. (1976) have suggested another oxidation pathway which may operate in, at least, rice bran. This pathway, based on isolation of some of the inter- mediates, would involve oxidation to a dioxindole and its ring expansion to a B-acid. At some point hydroxylation at indole ring carbon 5 would take place and the amide, ethanolamide and free acid of the hydroxy B-acid have been identified. The relative contribu- tion of this pathway to the degradation of the endogenous IAA in rice bran was not determined. Clearly the in viva degradation of IAA will be an important subject of future research, especially those as yet poorly understood pathways which do not decarboxylate and which apparently account for the bulk of the IAA which is degraded. 10 II. Environmental Effects on Indole—3-acetic Acid Levels The early work leading to the concept of hormone involvement in tropic behavior has been reviewed (Boysen-Jensen,l936; Went and Thimann, 1937; van Overbeek, 1939) and recently reexamined (Ehmann, l973). Duhamel du Monceau (1758), Knight (1806), De Candolle (1832) and Frank (1868, see also Rawitscher, 1932) observed and experimented with plant tropistic behavior in response to light and gravity. How— ever, it was Ciesielski (1872) and later Darwin (1880) who began the detailed study of the nature of the tropic responses. Ciesielski's methods, that is the removal of the tip and its replacement with the test material, has been the basis for many of the studies on tropic behavior and these methods are still in use (cf. Vanderhoef and Briggs, 1978; Vanderhoef et al., 1979). Ciesielski showed that removal of the root tip prevented growth and geotropism and he showed that replacement restored these properties. It was Blaauw (1918), however, who showed that phototropic bending was a function of the energy received by the plant and he correctly interpreted the tropic response as a result of differential growth. Boysen-Jensen (1910, 1913) showed the same effects for the light stimulus in coleoptiles as Ciesielski demonstrated for geotropism and further demonstrated that insertion of a thin piece of mica into a cut on the illuminated side did not prevent curvature but that insertion on the opposite side inhibited the phototropic response. If a block of gelatin was used in place of the mica, however, normal tropic bending occurred. Paal (1919) confirmed these results and showed that the substance 11 which could diffuse through gelatin but not through the mica sheet was also important in normal straight growth. Paal postulated, based on these experiments, that a substance moves from the tip and produces growth in the tissue below. In a series of 17 papers from 1918-1935 Cholodny (1924 and see review by Cholodny, 1935a) began to develop the concept of the mechanism of tropic behavior which, as refined by Went (1926), became known as the Cholodny-Went theory of tropisms. This theory stated that curvature of plant organs was a consequence of a light or gravity induced lateral diversion of auxin in the apical region of the organ. Since the amounts of auxin reaching the two sides of a rapidly elongating region would differ, the growth rates of the two sides would also differ and curvature would result. Evi- dence for the lateral diversion of auxin by geotropic induction was first provided by Dolk (1936), but whether a lateral redistri- bution of IAA is involved is still unclear. Briggs (1964) reviewed four theories for phototropic curvature which have played an important role in the understanding of phototropism. These theories are: (1) light induced changes in the tissue's ability to grow (perhaps in response to auxin (Blaauw, 1918)); (2) the light induction of lateral transport (Cholodny, 1935a); (3) the inactivation of auxin (Galston et al., 1953; Reinert, 1953; Brauner, 1953); and (4) the inactivation of some component of the auxin-synthesizing system (Galston, 1950, 1959). These same basic arguments have also been applied to geotropism, although the relationship between the two tropic 12 responses is still uncertain. Support for lateral transport in photo-induced plants has been provided by Went (1928), van Overbeek (1933), Asana (1938) and Wildin (1939) who used bioassay techniques to quantitate the auxin transported into receiver blocks at the bissected base of coleoptiles. However, the effect of light shown by these researchers was not only a redistribution of auxin but also a net decrease in total amount, possibly due to the red com- ponent in the white light used for illumination (Briggs, 1964). Although subsequent repetition of this work by Briggs et al. (1957) and Briggs (1963a) with better defined conditions has resulted in a smaller difference, this decrease is still unexpected since Went and Thimann (1937, also see Cholodny, 1929) indicate that total growth was the same in tropic stimulated and unstimulated coleoptiles. A more serious challenge to these early experiments came with the use of radiolabeled IAA to study tropism. Numerous groups (Bunning et al., 1956; Gordon and Eib, 1956; Reisener, 1957, 1958; Ching and Fang, 1958; Reisener and Simon, 1960; also see below) were unable to show a lateral redistribution of 14C-labeled IAA following either geotropic or phototropic induction. Pickard and Thimann (1964) were able to demonstrate a lateral redistribution of 14C—IAA both in agar receiver blocks and in the tissue, however only for the light exposures in the region of the first positive curvature were the data significant. Since the first positive curvature is morphologically limited and is a protracted response (Briggs, 1964) which is absent in coleoptiles of some species 13 (Asomaning and Galston, 1961), it is the region of the second posi— tive curvature which is somewhat more interesting. In those experi- ments with the second positive curvature Pickard and Thimann showed only a 46.4% distribution on the lighted side for oat coleoptiles and 46.1% for corn coleoptiles. Since they report recoveries of 14C-IAA of 98-106% it is safe to consider a redistribution of less than 8% to be insignificant. Shen-Miller and Gordon (1966) and Naqvi and Gordon (1967) used techniques similar to those employed by Pickard and Thimann except that they purified the IAA by solvent extraction and paper chromatography prior to counting and they bioassayed the radioactive fraction to confirm the distribution. The contribution of IAA resulting from hydrolysis of conjugates is difficult to estimate in these studies since they used cold ether extraction. However, the Shen-Miller and Gordon (1966) experiments show that, although the receiver blocks show a redis- tribution, an equal but opposite redistribution is found in the tissue. The sum of the IAA in the tissue and receiver block on the illuminated and shaded sides of the coleoptile is a constant. Thus, the results of these experiments are inconsistent with the theory of Cholodny-Went and suggest some mechanism which retards 14C—IAA movement through the shaded side of the coleoptile. Bruinsma et a1. (1975) used the fluorescence of the indolo-o-pyrone derivative to study endogenous IAA in sunflower 14 seedlings.2 Although this analytical method is as yet unverified by more rigorous methods, they were able to confirm earlier bioassay work which showed no lateral redistribution in this tissue (Blaauw, 1918; Went and Thimann, 1937). Wright et al. (1978) also used this analytical method to study IAA levels in grass nodes (see also early bioassay data of Schmitz, 1933). The findings of Wright et a1. indicate that although a differential is noted in upper and lower halves, the same distribution is obtained when the segments are split prior to geostimulation. Thus, in this tissue contiguity is unnecessary (see also Gradmann, 1925; Firn and Digby, 1977) and they attribute the effect to "gravity-controlled input evoking changes in IAA metabolism within small groups of cells of the leaf sheath base: perhaps an increase in rate of synthesis or decrease in the rate of degradation or conjugation of IAA in the lower sides with decreased synthesis or increased degradation or conjugation in the upper sides" 2Helianthus hypocotyls and, to a lesser extent grass nodes, have long been a source of data which conflicts with the Cholodny- Went theory of tropisms. Went and Thimann (1937, p. 161) note after describing the tropic behavior of Helianthus and nodes of grasses, that the result ". . . does not, however, agree with the Cholodny- Went theory . . ." and "It must be left for the present as an unexplained curiosity." Explanations for these inconsistencies involving gibberellins as the tropic hormone (Krass and Vardar, 1962; Phillips, 1972) have not been confirmed in subsequent work (Phillips and Hartung, 1976; Firn et al., 1977). It has been shown that only the outer cell layers of Helianthus hypocotyls respond to auxins (Mentze et al., 1977; Firn and Digby, 1977), which is not dissimilar from grass coleoptiles where the central cylinder is hollow. Many other dicotyledenous stems behave similarly (Cholodny, 1926; Iwami and Masuda, 1974; Mentze et al., 1977) and a hollow cylinder produced with a cork borer will exhibit normal tropic behavior. 15 (Wright et al., 1978). Clearly, while they were able to exclude the Cholodny-Went theory as an explanation they were unable, with the data they obtained, to distinguish between the synthesis and degradation theories (see above) and newer concepts involving the hydrolysis and formation of covalent linked conjugates for the con— trol of IAA levels and growth as illustrated by Bandurski et al. (1977) using careful analytical techniques. Wilkins' group used ”point source" applications of 5-3H-IAA to study redistribution of IAA during geotropic (Shaw et al., 1973) and phototropic (Gardner et al., 1974) induction. They found small redistributions in most experiments, but curvature was observed under conditions in which no lateral redistribution occurred. Also, they were able to confirm the observed light induced change in apparent rate of IAA transport reported by Shen-Miller and Gordon (1966). Phillips and Hartung (1976) failed to observe a gravity induced redistribution of 14 14 C-IAA in Helianthus, indeed they applied 2 uM of C-IAA in agar to the upper half of a Helianthus stem and obtained geotropic curvature while the radioactivity was distributed 4:1 upper to lower (i.e. the 14 C-IAA concentration was higher on the slower growing top half). It would seem, therefore, that although lateral transport of IAA may occur during tropic response it does not appear necessary under all conditions or in all tissues. Digby and Firn (1976) have suggested that the lateral redistribution noted by many workers may be due to IAA carried in the water which flows as the l6 growing cells expand.3 This hypothesis is supported by the experi- ments of Gardner et al. (1974) in which redistribution of 3H-IAA could only be observed in cut segments. Cut segments represent a closed system for water since little uptake from the agar occurs. Experiments with intact seedlings failed to show a redistribution of 3H-IAA, possibly because water was amply provided from below. It should be pointed out however that this area of research is one in which the use of careful analytical technique is woefully lacking. In none of the experiments described above can it be stated with certainty that the compound isolated or reisolated from the plant or agar block was in fact IAA. Only a few authors even carry characterization through a single chromatographic step (cf. Shen-Miller and Gordon, 1966; Shaw et al., 1973; Gardner et al., 1974) and a single chromatographic step does not prove identity. It will be important that future work carefully determine the identity and amounts of the compounds involved in tropisms if the biochemistry of tropic behavior is to be resolved. In addition to the tropic response, light affects plant growth several other ways. The most obvious of these is the change from the etiolated pattern of growth shown by seedlings raised in the absence of light to the light growth pattern. Characteristic of this change in growth pattern is inhibition of stem or coleoptile 3Goswami and Audus (1976) have shown that tropic stimuli also lead to a redistribution of various ions. This redistribution appears to be part of the response process rather than of perception as suggested by Cholodny (1922). 17 growth, a response which is closely related to phototropism differ- ing only in whether illumination is unilateral or equilateral (Elliot and Shen-Miller, 1976). This response has been found to be caused by very low intensities of blue light and has been defined as a light-growth response (Van Dilliwijn, 1925; also see Went and Thimann, 1937). Photo-inhibition of growth shows the same dose response curve as phototropism (Elliot and Shen-Miller, 1976; also see Briggs, 1960; Zimmerman and Briggs, 1963), the same change in sensitivity due to red light exposure (Elliot and Shen-Miller, 1976; also see Briggs, 1964) and similar spectral sensitivities (Elliott and Shen-Miller, 1976). In addition to these agreements between photoinhibition of growth and phototropism, a similar close correla- tive relationship exists between photoinhibition of growth and light inhibition of 14C-labeled IAA transport (Shen—Miller et al., 1969; Thornton and Thimann, 1967). If one accepts the rather convincing evidence that these responses are related, then the results of Pickard and Thimann (1964) that Show no effect of light on basipetal 14C-IAA transport are in contradiction with those of Thornton and Thimann (1967), Shen-Miller et al. (1969), and, as already discussed, in disagreement with the phototropic studies of Gardner et a1. (1974) and Shen-Miller and Gordon (1966). One possible explanation for an apparent decrease in rate of transport would be that light treatment results in an increase in the amount of conjugated IAA in the tissue. In time, the 14MM thus conjugated would be hydrolyzed and thus mask the apparent decrease in rate of 14C-IAA transport noted in the shorter time experiments (Figure 1). That this explanation is 18 .esmmPe use to seem easeme_ men ow xfiaaw xpco upsoz on ace Amy pamuxw mcvvcmn owaocpopoca cw csouo v_:oz cowpmzpwm «gem we» .Awuumowuec FepOp wgp new Auv a: vow: mew m>cmmwc mama uswcoo mo mpooa one we uwapocux; on szoz mummancoou<m -30; .mpcmewcmaxm pcoamcecp nmmcopoca mcwczo .<os cmzoem we“ op was Amv c? cmesecmewu we ucoqmcmcp ~eumawmmn mo can; esp cw mmwmcomv e m>cmmno n_:oz mpe>cmucw we?“ pcocm sew: asexcoz mpcwswcwaxm .<oE <epueowcmc Aza 2H ezsoz< museuzmhm ozaomzou 28 von Denffer et al. (1952) and these yielded IAA after mild treat- ments which suggested that they were close precursors of IAA. Stowe and Thimann (1953, 1954) suggested that a water soluble com- pound could be indolepyruvic acid, but this was disputed by subse- quent workers (Bentley et al., 1956; Britton and Housley, 1961) who showed that these compounds were alkali labile and chroma- tographically distinct from IAA and indolepyruvic acid. Additional chromatographic studies were carried out by Bennet-Clark and Kefford (1953) who described an "d-accelerator" which was appar— ently a neutral ester fraction which they extracted from corn as well as several other plants. Similar studies were made by Farrar et a1. (1958) whose active fraction was probably the inositol esters of corn seed discussed below. This early progress has been reviewed by Stowe (1959) and Bentley (1958, 1961). Hamilton et al. (1961) were the first to quantitatively show that the esters of IAA in corn kernels consisted of about half water soluble and half water insoluble (high molecular weight glucan, see above) compounds. Labarca et al. (1965) partially character- ized a series of major water soluble esters of IAA from kernels of Zea mays. Four chromatographically distinct compounds were found (Bl-B4), two (B1 and B2) which contained IAA and inositol and were interconvertible. The other spots (B3 and B4) contained arabinose and could be converted to the B1 and B2 esters by snail gut juice (mixed glucosidases). An earlier report of IAA-arabinose (Shantz and Steward, 1957) in corn seed was, probably, an incorrect identi- fication of the B3, B4 compounds. The identity of the IAA-inositol 29 conjugates was confirmed by Nicholls (1967) who identified the 82 compound as indole-3-acetyl-2—0—myo—inositol by comparison with a synthetic standard and by its characteristic downfield chemical shift (6) in the nuclear magnetic resonance spectrum (Nicholls et al., 1971). Ueda et al. (1970) identified an additional ester as containing IAA, myo—inositol and galactose and Ueda and Bandurski (1974) established the structures of the IAA-inositol ester com— pounds in a comprehensive coupled gas chromatography-mass spec- trometry (GC-MS) study. Ueda and Bandurski (1974) also showed that galactose and arabinose were 5-0-pyranosyl on the IAA—myo—inositol glycosides. Ehmann (1974a) established the identity of 2,4 and 6-0 esters of IAA and glucose and Ehmann and Bandurski (1974) estab— lished the identity of IAA-nyo-inositol esters with two and three molecules of IAA esterified to the cyclitol. Thus, all of the IAA containing compounds of Zea mays kernels present in amounts of 10 ug/kg or greater have been identified and their concentrations determined (Bandurski et al., 1969; Ueda and Bandurski, 1969; Ehmann, 1973) as summarized in Table l. The formation of these compounds in developing kernels and their decrease in amount during germination has been described (Corcuera, 1967; Ueda and Bandurski, 1969). Piskornik (1975) has also shown that the indole compounds of corn kernels occur almost exclusively in the endosperm. The in vitro enzymatic synthesis of IAA-myo-inositol and IAA—glucose has been described (Kopcewicz et al., 1974), further characterized as to its cofactor requirements (Michalczuk and Bandurski, 1979) 30 and the chemical synthesis of 14 C-IAA-myo—inositol has been accomplished (Nowacki et al., 1978). No other plant material has been so carefully studied as to its bound auxin content as Zea mays kernels, however several other esters of IAA have been partially described. Percival and Bandurski (1976) described an ester of IAA from Avena seed which accounted for about 80% of the IAA found in this seed. This ester was found to be a heterogenous glycoprotein of 5,000 to 20,000 daltons and the carbohydrate was a mixed 8 1-3, 1-4 lichenin type glucan. Avena also contained small amounts of IAA which was liberated by 7 N NaOH at 100 C for 3 hrs and was presumably in amide linkage to some other compound. Although their analytical methods were inadequate, Kaglevic and Pokorny (1969) reported cochromatography of a compound from Avena coleoptiles with synthetic 1-0-indole-3-acetyl-B-D-glucopyranose and Shantz and Steward (1968) partially characterized an ester of IAA which yielded rhamnose and glucose which was isolated from immature fruits of Aesculus woerlitzensis. IAA and rhamnose were suggested by chromatography as the hydrolysis products of a compound isolated by Ganguly et al. (1974) from floral parts of Peltophorum ferrugineum. Several authors have reported chromatographic evidence for the amide linked conjugate indole—3-acetyl-L—aspartic acid as a natural product (Klambt, 1960; Row et al., 1961; Olney, 1968; Tillberg, 1974) but confirmation is still necessary by more rigorous physical techniques. Sircar and Das (1951, 1954) and Sircar and Chakravorty (1957) con- ducted early bioassay studies of the bound IAA of rice. Their 31 results (Sircar and Das, 1954) show a decline in bound IAA with time after germination similar to that shown by Ueda and Bandurski (1969) for corn using chemical methods. Removal of the embryo from the germinating grain prevented this decline in amounts. In agree- ment with the data of Piskornik (1975) with corn, bioassay data indicate that rice contains 80% of its total IAA in the endosperm (Sircar and Chakravorty, 1957). Recently, one of the bound forms of IAA in rice has been identified as indole-3-acetyl-myo-inositol by cochromatography of the intact compound and its hydrolysis pro- ducts on thin layer, high pressure liquid, and gas chromatography as well as by combined GC-MS (Hall and Bandurski, 1979). Bandurski and Schulze (1974, 1977b) have quantitatively studied the IAA con- tent of a number of plant tissues including rice and, based on the alkali lability of the IAA compounds, have classified them as free, ester and amide forms. The biological activity of a variety of halogenated indoles has been examined (Stevens and Fox, 1948; Hoffmann et al., 1952; Sell et al., 1953; Porter and Thimann, 1965) and it was found that substitution with chlorine in the 4 position of IAA resulted in a compound with up to 700 percent of the biological activity of the parent compound. The molecular implications of this activity enhancement has recently been reexamined (Katekar, 1979) and the biological activity in several additional bioassay systems has confirmed the high activity of 4-chloro-indole-3-acetic acid (Marumo et al., 1974; Bottger et al., 1978b). The methyl ester of 4-chloro-IAA has been isolated and well characterized from 32 immature pea seeds by several groups (Marumo et al., 1968a; Gandar and Nitsch, 1967; Engvild et al., 1978) and the free acid has also been reported (Marumo et al., 1968b). The bulk of the 4-chloro-IAA is as the methyl ester (87 pg/kg) with the free acid accounting for only 14 ug/kg. The mono methyl ester of 4-chloro-indoly1-3-acetyl- L—aspartate has also been identified by rigorous chemical methods from inmature pea seed and accounts for 9 pg/kg (Hattori and Marumo, 1972). Interestingly, Hattori and Marumo (1972) were unable to isolate IAA or IAA methyl ester from this plant material by methods 'which should have detected as little as 4 pg/kg. Although previous bioassay data had claimed IAA isolation (Housley and Griffiths, 1962), this was probably due to an artifact of the isolation methods which do not separate IAA from 4—chloro-IAA (Marumo et al., 1971). Mature pea seeds, however, contain IAA (93 ug/kg), an amide linked conjugation product (202 ug/kg), but no esterified IAA (Bandurski and Schulze, 1977b). This conversion from halogenated to primarily non-halogenated indole metabolism in the developing pea seed must be studied further. Redemann et a1. (1951) isolated a compound from immature corn kernels which appeared to be the ethyl ester of IAA. The use of ethanol in isolation makes this uncertain since esterification of the free acid can occur during extraction and the report was subsequently withdrawn (Fukui et al., 1957). If ester linked con- jugates were present, transesterification to the alcohol could also result, as reported by Zenk (1964). A similar criticism could be applied to the methyl esters of the chlorinated compounds (see 33 above) since in all cases the investigators used methanol for extraction. Marumo et al. (1968b) did utilize an acetone control, but their analysis of this control by bioassay was insufficient evidence for the endogenous nature of these methyl esters. A recent report (Hofinger and Bottger, 1979) using alkaline buffer extraction, however, confirms the presence of the methyl ester. The methyl and ethyl esters of IAA are potent stimulators of parthenocarpy (Sell et al., 1953). Seeley et a1. (1956) presented chromatographic evidence of methyl ester hydrolysis by several plant tissues. Conjugates of IAA from non-plant sources have been reported. Zenk (1960) isolated an enzyme from liver mitochondria which syn- thesized indole-B-acetylglycine via an IAA-CoA intermediate. Indole-3-acetylglucosiduronic acid (Jepson, 1958) and indole-3- acetylglutamine (Jepson, 1956) have been isolated from abnormal human urine. A plant pathogenic bacterium, Pseudomonas savastanoi, produces indole—3-acetyl-e-L-1ysine (Hutzinger and Kosuge, 1968a) in large amounts (Hutzinger and Kosuge, 1968b). Also, numerous plant indole conjugates have been reported which are not IAA derivatives. For example, Ehmann (1974b) isolated N(p-coumaryl)- tryptamine and N-ferulyl-tryptamine from corn kernels and the corresponding 5-hydroxy compounds as well as the B-D-glucopyranoside have been isolated from Carthamus tinctorium (Sakamura et al., 1978). A stigmasteryl-B-D-glucoside has been reported from peanut plants and has a synergistic effect with IAA in Avena elongation bioassays (Kimura et al., 1975). The physiological significance 34 of these conjugates is unknown, however the urine compounds might be considered excretion products and the lysine conjugate could play a role in a pathogenic mechanism. In addition to the natural products discussed above, a number of conjugates have been identified as products of exogenous auxin application. Andreae and Good (1955) identified indole- acetylaspartic acid as the major metabolite of pea stems and roots fed with large amounts of IAA. Andreae and Good (1957) extended these studies to a number of other indoles and auxins. Indole- propionic acid, indolebutyric acid, 2,4-dichlorophenoxyacetic acid (2,4-D) and benzoic acid gave products with properties identical to those of the respective synthetic aspartic acid derivatives. Sub- sequent reinvestigation however revealed that the benzoic acid derivative was benzoylmalic acid (Venis and Stoessl, 1969). Trace amounts of the aspartic acid conjugate were formed in tissue which had been pretreated with auxins, but none was produced in tissue not pretreated. Thus, this is an example of the errors possible when identification is based simply on chromatographic migration of impure extracts. Zenk (1962; also, cf. Schraufolf, 1971) reported the formation of the aspartic acid conjugate with the additional synthetic auxin, napthaleneacetic acid. The ability of the plant tissue to make the aspartate con- jugates is enhanced by pretreatment with a variety of active auxins (Andreae and Van Ysselstein, 1956; Sudi, 1964; Venis, 1964; Sudi, 1966; Venis, 1972) such that pretreatment with IAA will lead to increased ability to conjugate the synthetic auxins as well as IAA. 35 Aside from this apparent inducibility, this enzymatic activity has not been further characterized except for two reports of in vitro synthesis (Lantican and Muir, 1969; Higgins and Barnett, 1976) which have been disputed (cf. Venis, 1972). Although the work cited above was done with peas, Good et al. (1957) and Zenk (1964) showed that a large number of plant tissues would produce compounds which were chromatographically similar to the aspartate conjugate when exogenous IAA was applied. Klambt (1961) and Zenk (1961) isolated a compound after exogenous application of IAA which appeared to be the glucose ester of IAA and to which they assigned the structure l-O-indole—3—acetyl— S-D—glucose. Adequate published chemical documentation of the 1-0 linkage is still lacking. Several years earlier the B—glucosidic ester of indole—3—propionic acid had been identified as a product of cultures of Bacillus megatherium (Tabone and Tabone, 1953). The presence of the glucoside in treated higher plants suggests that the indoleacetamide found on chromatograms by Good et a1. (1956) was a product of ammonolysis with the solvents employed (Jepson, 1958). Synthetic 1-0-indole-3-acetyl—B-D-glucose has been reported to have a higher biological activity than IAA (Keglevic and Pokorny, 1969), however this may be a result of various factors such as uptake rate. Zenk (1964) speculates that the IAA-glucose compound is a transient form which is made prior to the appearance of the enzymatic activity which produces the aspartate conjugate. A comparison of the data of Good et al. (1956) with that of Bandurski and Schulze (l977b) indicates that those plants which make large amounts of ester 36 conjugates are those with a high endogenous amount of ester IAA (see also data of Feung et al., 1978 for 2,4-D). Therefore, it is more likely that the ratio of complexes formed from exogenous appli- cation is species related as proposed by Good et a1. (1956). It is also difficult to envision how a complex multiproduct response as proposed by Zenk (1964) might evolve in plants when environmental exposure to large amounts of exogenous auxins was an unusual occur- rence until the advent of modern growth regulators. ‘Feung et a1. (1976) identified IAA conjugation products in tissue culture from Parthenocissus crown gall by chromatographic and mass spectral techniques. They identified the glycine, alanine, and valine conjugates as major products and the aspartate and gluta- mate conjugates were formed in lesser amounts. Feung et a1. (1977) also studied the biological activity of 20 L-a-amino acid conjugates of IAA in Avena straight growth and in supporting growth of soybean cotyledon tissue culture. Recently, interest in these compounds for control of morphological development in tissue culture has developed (Peterson, 1978; Hangarter et al., 1979). Feung et al. (1974) also studied the biological activity of the 2,4-D amino acid conjugates and numerous other reports exist on various biological activities of one or more IAA conjugate (cf. Jerchel and Staab-Muller, 1954; Nicholls, 1967; Keglevic and Pokorny, 1969; Rekoslavskaya and Gamburg, 1976). Because of their wide usage as herbicides the metabolism of the phenoxyacetic acids has been carefully studied. Holley (1952) found that a major metabolite of 2,4-D appeared to be a ring 37 hydroxylated glycoside. Thomas et al. (1964b) confirmed this and showed that the hydrolysis products cochromatographed with 2,4- dichloro-5-hydroxyphenoxyacetic acid and, as a minor component, 2,3-dichloro—4-hydroxyphenoxyacetic acid. In contrast to the results with bean plants, oat seedlings appeared only to produce the glucose ester (Thomas et al., 1964a). This earlier work on the carbohydrate containing auxin conjugates was the subject of a review by Hilton (1966). As discussed above, Andreae and Good (1957) found chromatographic evidence for the formation of 2,4-dichlorophenoxy— acetylaspartic acid. This was confirmed, again only by chromato- graphic migration, by Klambt (1961) who also provided evidence for the glucose ester. Hamilton et al. (1971) confirmed the presence of the hydroxylated phenoxyacetic acids in treated bean plants using mul- tiple chromatographic systems as well as mass spectrometry. They found the major products of 2,4-D treatment to be 2,5-dichloro-4- hydroxyphenoxyacetic acid, while 2,3-dichloro-4-hydroxyphenoxyacetic acid was a minor metabolite. These compounds accumulated as the glycosides which could be freed to the aglycone by a commercial B-glucosidase. Results with soybean cotyledon callus tissue showed that they yielded the same products as well as a conjugate of 2,4-D with glutamic acid (Feung et al., 1971). Feung et a1. (1972) identified the aspartic acid conjugate and confirmed this, as well as the structure of the glutamic acid metabolite, by mass spectral data. Arjmand et al. (1978) found the aspartate and glutamate conjugates to be the major products of soybean callus supplied with 38 2,4,5-trichlorophenoxyacetic acid. Additional studies (Feung et al., 1973b) characterized five amino acid derivatives of 2,4-D: alanine, valine, leucine, phenylalanine, and tryptophan. In addition, they found that callus fed with the glutamic acid conjugate of 2,4-D pro- duced the aspartic acid conjugate in larger amounts than tissue supplied with the free acid. Davidonis et al. (1978) have shown that in very young root callus from soybean that the levels of free 2,4-D increased with amount added but that the levels of glycosides and amino acid conjugates increased only slowly. This was con- trasted with the situation in more mature callus in which the free 2,4-D concentration remains at a constant, apparently regulated, level. This level is maintained by the tissue by increasing its rate of 2,4-D conjugation as the levels of added 2,4-D are increased. Thus, the older root callus appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram of tissue, and this was pri- marily by formation of the amino acid derivatives. Feung et al. (1973a) published an extensive mass spectral and chromatographic study of twenty L-form amino acid conjugates of 2,4-D and Feung et al. (1975) published a similar study on amino acid conjugates of IAA. These will serve as important reference spectra for future work on these compounds. B. Gibberellin Conjugates Murakami (1961) found that cucumber leaf disks produced large amounts of gibberellin B-glucoside when floated on solutions of gibberellin A3. Although the product was not well characterized, 39 this glucoside of A3 had chromatographic and partition properties similar to the water-soluble, non-ethyl acetate extractable gibberel- lins described as natural products by later investigators (Murakami, 1962; Ogawa, 1963; Zeevaart, 1966). Hashimoto and Rappaport (1966) showed that developing bean seeds exposed to gibberellin A1 converted most of it to water-soluble compounds and that the biological activ- ity of the gibberellins in the solvent phase remained constant regardless of the dosage of applied gibberellin A]. Barendse et al. (1968) showed using 3H—gibberellin A1 that a compound was formed in developing Pharbitis and pea seeds which would yield gibberellin A1 upon mild acid hydrolysis and that this compound was also converted to 3H-gibberellin A1 during seed germination. Although Barendse et al. did not characterize the "X" products found in the seeds, Murakami (1968) showed that Pharbitis seeds contain a water-soluble, biologically inactive compound which could be converted to an active gibberellin by hydrolysis with 0.2 N H2504. Tamura et al. (1968) isolated three water-soluble neutral gibberellins from Pharbitis seeds. The major components accounted for 50 ug/kg out of the total of 72 pg/kg of neutral gibberellins present. Based on the chemical properties of the hydrolysis pro- ducts and the nmr spectra of the intact compound, it was assigned the structure 2-0—B—glucosyl-gibberellin A3. Schreiber et a1. (1967) identified the related 3-0-B-D-glucopyranosyl-gibberellin A8 from seeds of Phaseolus coccineus. These two publications were the first reports on the isolation and characterization of endogenous glysosidic gibberellins. Schreiber et a1. (1969) later confirmed 40 the identity of the glycoside by synthesis and thus established the structure of the compound they had described years earlier (Sembdner et al., 1964) as the "Phaseolus a" compound based on solvent partitioning and chromatography. Yokota et al. (1969) described additional glycosides from Pharbitis and their chemical characterization was covered in an extensive publication which appeared later (Yokota et al., 1971). These compounds, which accounted for most of the gibberellin in the seeds, were: 2-0-B-glucosyl-gibberellin A3 (as above); 2-0-8- glucosyl-gibberellenic acid; 2-0-B-glucosyl-isogibberellin A3; 3-0-B-glucosyl-gibberellin A26; 3-0-B-glucosyl-gibberellin A27; 3-0—8-glucosyl-gibberellin A29; and 3-0-B-glucosyl-gibberellin A8. The 3-0-B-glucosyl-gibberellin A8 was also isolated from shoot tips of Althaea rosea (Harada and Yokota, 1970) where the relative levels of free gibberellin and glucoside appeared to correlate with shoot development. Yamane et a1. (1974) characterized a glucoside of gibberel- lin A35 from Cytisus scoparius which upon careful chemical analysis proved to be ll-O-B-D glucosyl gibberellin A35. Hiraga et a1. (1972, 1974b) isolated four glucosyl ester compounds which were identified as esters of gibberellins A], A4, A37 and A38. The ester forms were present in mature Phaseolus seeds but absent in immature seeds. Lorenzi et a1. (1976) isolated and characterized the gibberellin A9 glucosyl ester from Picea sitchensis and Yokota et al. (1975) published a mass spectral study of the glucosides and ester compounds. Evidence for a high molecular weight bound 41 gibberellin was provided by Halinska and Lewak (1978) although they did not characterize the intact compound. The 2-0-acetyl gibberellin A3, the only non-glucose containing natural conjugates so far identified, was isolated from Fusarium (Schreiber et al., 1966). The biological activity of these compounds has been studied (Yamane et al., 1973; Hiraga et al., 1974a; Sembdner et al., 1976) but other aspects of the biology of these compounds have only been suggested. Sembdner et a1. (1968) state that these compounds may function as "depot" forms in the seed which are used during germi- nation since conjugated gibberellins form during maturation of fruits and yield the free compound during germination (Sembdner, 1974). Likewise, they suggest a long distanct transport role based on the presence of conjugated gibberellins in the bleeding sap of Acer platanoides and Ulmus glabra (Sembdner et al., 1968). Also, several authors have suggested a role for these compounds in seed dormancy (cf. Halinska and Lewak, 1978) and the results of Hashimoto and Rappaport (1966) suggest the possibility of a system for mainte- nance of free hormone levels similar to that shown with IAA and 2,4-D (Bandurski et al., 1977; Davidonis et al., 1978). Further work on the physiology and biochemistry of these compounds should, therefore, lead to new insights into the regulation of gibberellin levels and how gibberellins regulate plant processes. 42 C. Cytokinin Conjugates Cytokinins, like the phytohormones IAA and gibberellins discussed above, exist in plant tissue in both bound and free form. Zwar et al. (1963, 1964) isolated several active fractions of cyto— kinins from apple and from coconut milk and found that fraction IV could be converted to fraction II by mild acid hydrolysis. These results were confirmed by Loeffler and van Overbeek (1964) and Wood (1964) reported the partial characterization of a cytokinin gluco- side from crown gall cultures of Vinca rosea. Kende (1965) showed a similar change in chromatographic behavior following acid treat- ment of a fraction from sunflower root exudates and suggested a role for these compounds in hormone translocation. Yoshida and Oritani (l972) isolated a putative zeatin glucoside from rice, par- tially characterized the compound and suggested it was zeatin-9—B- glucoside. This identification was, however, in error since synthetic zeatin-9-B-D-glucoside is not hydrolyzed by B—glucosidase (Parker et al., 1975) and their compound was labile to this enzymatic hydrolysis. Van Staden (1976) claimed the identification of a zeatin glucoside from coconut milk, however the procedures utilized were insufficient to establish the position of the linkage and the identification of the hydrolysis products is uncertain. In experiments where cytokinins have been fed to plant tissue several products have been identified. For example, Deleuze et al. (1972) identified the 7-glucoside of 6-benzylaminopurine in several plant systems after treatment with the parent compound and, based on its mass fragmentation pattern, assigned it the 43 glucofuranose configuration. Additional metabolites have also been identified as benzylamino-9-B-D-ribofuranosylpurine and its 5'- monophosphate (Fox et al., 1972; Dyson et al., 1972) and the 3 and 9-glucosides of 6-benzyl-aminopurine (Wilson et al., 1974; Letham et al., 1975). Similarly, treatment of plant material with zeatin (6-(4-hydroxy-3-methylbut-trans-Z-enyl-amino)-purine) produces metabolites identified as 7-glucosylzeatin, 9—glucosylzeatin, zeatin riboside and its 5'-monophosphate, and dihydrozeatin (6-(4-hydroxy- 3-methy1butylamino)purine) and its riboside and riboside 5'-phosphate (Sondheimer and Tzou, 1971; Parker et al., 1972; Parker and Letham, 1973; Parker et al., 1973; Tzou et al., 1973; Parker and Letham, 1974; Duke et al., 1979). Several zeatin metabolites with a 4— glucosyloxy moiety have also been identified by Letham et a1. (1976) as O-B-D-glucopyranosyl-9-B-D-ribofuranosy1dihydrozeatin, O-B-D- glucopyranosylzeatin, O-B-D-glucopyranosyldihydrozeatin and O-B-D- glucopyranosyl-cis-zeatin. The formation of the nucleotides of the 6-furfuryl-, 6—methy1-, and 6-propy1-aminopurines was shown by Doree and Guern (1973) who also found none of the corresponding nucleo- sides. Recently, a detailed study of the chemical synthesis and properties of many of these compounds has appeared (Cowley et al., 1978) and also a preliminary report has been published on their enzymatic synthesis (Entsch and Letham, 1979). Several endogenous cytokinin conjugates have been identified with a precise chemical structure. Miller (1965) and Letham (1966a, b) described the compounds 9-B-D-ribofuranosylzeatin and its 5'- monophosphate from immature sweet corn and the riboside was also 44 convincingly shown to be present in the liquid media of a culture of Rhizopogon roseolus (Miller, 1967). Hall et al. (1967) identi- fied the cis isomer of zeatin riboside (6-(cis-4-hydroxy-3- methylbut—2-enylamino)-9—B-D-ribofuranosylpurine) from immature sweet corn and Horgan et al. (1973) isolated a novel cytokinin, 6(0-hydroxybenzylamino)-9—B-D-ribofuranosylpurine, from Populus robusta leaves. Wang et al. (1977) found 6-(4—0-8-D-glucosyl-3- methylbutylamino)purine, a dihydrozeatin glucoside, in leaves of Phaseolus and identified it by a variety of chemical and biochemical tests including combined GC-MS. Peterson and Miller (1977) and Morris (1977) elegantly identified two cytokinin glucosides from cultures of Vinca rosea crown gall which, earlier, Wood (1964) had studied but not fully characterized. These compounds were a glucosylzeatin (6(4-0-8—0-91ucopyranosyl-3—methyl-trans-but—2- enylamino)purine) and a glucosyl zeatin riboside (9—B-D—ribo- furanosyl-6(4-0-B-D-glucopyranosyl-3—methyl-trans-but-2-enylamino) purine). The naturally occurring cytokinin 6-(3-methyl-2-butenyl) aminopurine is converted to adenosine by an enzyme from tobacco tissue cultures (Paces et al., 1971) and Lemna minor metabolyzes 6-benzylaminopurine to adenine (Bezemer-Sybrandy and Veldstra, 1971). Similar reactions have been observed to be catalyzed by mammalian xanthine oxidase (E.C. 1.2.3.2) acting on 6—(furfurylamino)purine (Bergmann and Kwietny, 1958; Henderson et al., 1962) and the sub— strate specificity of this type of reaction catalyzed by an enzyme from immature Zea mays kernels has been studied by Whitty and 45 Hall (1974). The enzyme is equally active on naturally occurring cytokinins and their ribosides, but cytokinin analogs with satu- rated or bulky side chains do not react. Thus, Wang et a1. (1977) have suggested that the function of the dihydrozeatin glucoside which they isolated from bean might be related to the compound's resistance to such oxidative reactions. Although careful quantitative studies have not, apparently, been done on the relative amounts of the various cytokinin deriva- tives in plants, numerous studies based on bioassay indicate that the conjugates account for more than half of the cytokinin activity of various plant extracts. This value is probably low, however since the glucosides are not as active as the free compound in some bioassays (Van Staden and Papaphilippou, 1977) and because of the lability of the compounds, especially in extracts where enzymatic hydrolysis has not been carefully eliminated. A brief report on germinating seeds indicate that the bound forms may serve as seed reserves since the glucosides were only found in the endosperm and declined after germination (Davey and Van Staden, 1977). Julin- Tegelman (1979) found that zeatin ribotide decreased during germi- nation in Zea mays but not in sufficient amounts to account for new cytokinin which appeared. Though Julin-Tegelman suggested that these results indicated de novo synthesis, the possibilities of other bound cytokinins such as the glucosides was not examined. 46 D. Abscisic Acid Conjugates Osborne (1955) first published evidence for the existence of diffusable abscission-accelerating substances. Such a compound was isolated and characterized by Liu and Carns (l96l), Ohkuma et al. (1963), and Cornforth et al. (1965) and the structure 3-methyl-5-(l'-hydroxy-4'—oxo-2'~cyclyhexen-l'-yl)-cis—2,4- pentadienoic acid was confirmed by synthesis by Cornforth et al. (1965) and Roberts et a1. (1968). This compound has been given the trivial name of abscisic acid (Addicott et al., 1968) although early workers referred to it as abscisin, abscisin I, abscisin II and dormin. Koshimizu et a1. (1968) isolated (+)abscisyl-B-D-gluco- pyranoside from the immature fruit of Lupinus luteus and confirmed its structure by exacting physical methods including nmr and mass spectrometry, thus establishing the structure of the first naturally occurring bound form of abscisic acid. Milborrow (1968) found that 14C-abscisic acid fed to bean the major metabolite of racemic petioles or Acer pseudbplatanus sections was a water soluble com- pound later shown (Milborrow, 1970) to be identical to that isolated by Koshimizu et a1. (1968). The compound has now been synthesized by preparation of the 2,3,4,6-tetra-0-acetyl-B-D-glucopyranosyl ester followed by an enzymatic deacetylation (Lehmann et al., 1975). Milborrow (1970) also found that trans-trans-abscisic acid was con- verted to its glucose ester 10 times faster than the cis-trans isomer and several laboratories have found that additional free absicsic acid is released by alkaline hydrolysis of methanolic 47 plant extracts (Milborrow, 1968; Rudnicki and Pieniazek, 1971; Osborne et al., 1972; Goldschmidt et al., 1973; Milborrow, 1974). Most of these reports find that the forms released by hydrolysis account for one third or less of the abscisic acid in the tissue. Again, these values must be viewed as lower limits since careful quantitative work has not been done and in at least one study (Goldschmidt et al., 1973) the "bound abscisic acid" exceeded the free acid by ten fold. Leshem et a1. (1974) Showed a change in the amounts of free abscisic acid and its glucoside during bud dormancy break in almond. Using a gas-liquid chromatographic assay system they were able to detect a decrease in free abscisic acid, including the cis-trans and trans-trans isomers, and an increase in the glucoside over a 15 week period. These data were confirmed by the later work of Wright (1975) and Harrison and Saunders (1975) on dormant buds of various deciduous plants. Barthe and Bulard (1978) examined abscisic acid levels in dormant and after-ripened embryos of apple using both bioassay and gas-liquid chromatography. Previously, they had found abscisyl-B-D-glucopyranoside in apple embryos (Bulard et al., 1974) by gas chromatographic identification of the hydrolysis products. They found a spectacular decrease of the free abscisic acid level following release from dormancy and a parallel increase in the bound form which became the major form. These data extended the earlier data of Rudnicki (1969). Hiron and Wright (1973) found that plants subjected to water stress had increased levels of abscisic acid and that as the abscisic acid levels dropped during «‘5: 48 recovery, increased amounts of conjugated abscisic acid could be detected. Thus, they postulated that conjugate formation was a slow adaptive mechanism which prevents the rapid reopening of stomates following water stress. The conjugation of plant hormones appears to be a general phenomenon in plant tissue, both in the fact that all plants so far critically examined have been found to have one or more conjugated hormone and that all of the major plant hormones (auxins, gibberel- lins, cytokinins and abscisic acid) form one or more conjugate. Many similarities in physiology appear among the conjugates of various hormones, although work on many of them is still in its infancy. Several have been suggested as transport forms, as pro- tectants against oxidative attack, as regulators of hormone levels, and as slow release forms. Clearly, no future work on plant hor- mones will be complete without considering this important aspect of hormone metabolism and studies which examine the interactions between multiple hormones, both bound and free, could provide new insight into the hormonal relationships of higher plants. A general review of plant hormone conjugates, with a more Germanic viewpoint, has appeared (Sembdner, 1974). 49 IV. Analytical Chemistry of Indole-3-acetic Acid and Its Adducts Indole chemistry began in the mid-nineteenth century with investigations on the dye indigo, which had been highly valued since ancient times. Baeyer and Emmerling (1869) proposed the presently accepted formula of indole, a benzopyrrole in which the benzene ring is fused to the 2- and 3-positions of the pyrrole ring. Several chemical aspects of the structure of indoles have direct bearing on its analysis. Indoles have a planer structure with ten n-electrons free to circulate throughout the molecule, two of which originate from the nitrogen atom thus making them relatively unavail- able for salt formation as compared to those of, for example, pyri- dine. Indoles belong to the group of heterocycles designated n- excessive heteroaromatics (Albert, 1959), since n-electron densities on indole carbons are greater than those of benzene. Thus, they are highly reactive towards electrophilic reagents, including acids and some oxidants (Remers, 1972). The nitrogen of indole is relatively acidic with a pKa of 17, such that anion formation occurs only in strong base (Remers, 1972). The anion increases reactivity of the C(3) to electrophiles, but the ring system itself is relatively base stable. Indoles are strongly ultraviolet (U.V.) absorbing with IAA having peaks at 220-222 nm (log e=4.51) and 279-282 nm (log c=3.78) and shoulders at 273 nm (log e=3.77) and 294 nm (log e=3.7l) (Hinman and Lang, 1964; Bandurski and Schulze, 1974). They are also strongly fluorescent with the excitation maximum for the IAA 50 anion at 292 nm, the emission at 362 nm and a quantum efficiency of 56.5% (Bridges and Williams, 1968). Adsorption of a photon results in a pronounced shift in the pKa of the indole, calculated to be 7.5 units (Longworth et al., 1966), making the molecule more prone to oxidative attack. At 77° K in rigid ethanol glass IAA will exhibit phosphorescence with excitation at 285 nm and emission at 436 nm and a Te of 7.1 seconds (time required for a decrease in intensity to 36.8% of initial) (St. John et al., 1967). These chemical properties place certain intrinsic con- straints on the quantitative analysis of microsamples of IAA. Because of the ease of indole oxidation, especially when exposed to U.V. light, and the propensity of the planar ring to adhere to glassware, large and variable losses occur during isolation. Moore and Shaner (1967) found 30% losses each time paper chromatography was performed and Little et a1. (1978) have reported losses of over 99% during isolation. Hamilton et a1. (1961) found that recoveries of IAA were between 0 and 56%, with the lower yields occurring with plant tissue or radioactivity present. Mann and Jaworski (1970) studied methods for minimizing losses during IAA isolation and found, as expected, that oxygen, light and protracted extraction times resulted in lower yields. Under their conditions using low actinic glassware, antioxidants and nitrogen atmosphere yields of 60% were obtained, although they used plant material fortified with unphysiologically high amounts of IAA. Clearly, any method for the isolation of IAA from plant material which does not use an internal standard to measure losses during sample preparation is unreliable. 51 Unfortunately, such corrections are rarely used and the first use of isotope dilution methods (Rittenburg and Foster, 1940) for IAA analysis was not until 1960 (Turian and Hamilton, 1960) even though 14C-labeled IAA was available much earlier (cf. Stutz et al., 1951). Detection of IAA in plant tissues requires separation of an IAA containing fraction from the bulk of the cell debris. Early workers used five basic methods (Boysen-Jensen, 1936): (l) diffu- sion of growth substance into agar or dextrose agar, (2) diffusion into water, (3) extraction with alcohol, (4) extraction with chloroform and (5) extraction with water. Because of problems with autolysis of bound IAA in tissue extracted with highly non-polar solvents or with water (Hamilton et al., 1961), most workers now use aqueous alcohol (cf. Hamilton et al., 1961; McDougall and Hillman, 1978a) or acetone (cf. Ueda and Bandurski, 1969; Bandurski and Schulze, 1974) for extraction. For some special situations the use of 80% (NH4)ZSO4 (Atsumi et al., 1976) or alkaline buffer (Hofinger and Bottger, 1979) extraction have proven useful but the use of aqueous acetone is advantageous since problems with esteri- fication during extraction, as discussed previously, are avoided. Methods of trace enrichment, as applied to replace extraction and solvent partitioning in the purification of the cytokinins (Kaiss- Chapman, 1977), have so far not been successfully applied to the indole hormones. Chromatographic purification of IAA was first carried out by Yamaki (1950) who applied paper chromatography to study the auxins in plant material. Since that time a variety of paper, 52 thin layer, column, high pressure and gas-liquid chromatographic systems have been applied to the purification of IAA. Paper and thin layer chromatographic methods have been the most widely employed because of the sensitivity afforded and probably because the equipment required is minimal. Some of the common paper and thin layer chromatographic solvent systems employed for IAA puri— fication are summarized in Tables 2 and 3. Unfortunately, paper chromatography for the purification of IAA is less well documented than thin layer and fewer than 30% of the authors cited who employed this technique provided relative mobility data and many did not even state the source of the paper utilized. Paper electrophoresis has also been applied to IAA by some workers (Hamilton et al., 1961; Hemberg, 1972). Although still useful, these methods have the prob- lem of exposing the compound to large surfaces where oxidation may result. Because of its higher resolution, silica gel thin-layer chromatography minimizes this exposure, despite the fact the support is acidic. Thus, recoveries are generally low with these methods and for critical work at submicrogram levels they can only rarely be employed. A variety of column chromatographic methods have been used for IAA isolation. Silica gel and diatomaceous silica found early appeal (Hamilton et al., 1961; Seeley and Powell, 1964; DeYoe and Zaerk, 1976) and still find utility where relatively large amounts of plant material are to be analyzed. Indoles chromatograph on a variety of polymeric materials, probably because of their planar structure. This property has allowed the use of various Sephadex 53 Awmmpv aconEo: Axomv Focwcpm comma Ammmrv oc=meoz use w¥o5o> . 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C NC Cm_ mmaz .ozNC o:-<C Nm._ meme\Ccv CCN1CNP <m CCC coeemcm Cm CC1mm um._ CC, ewe + :CCm1PC cC 1Com A_C C1C+m Nm1<m CCC coemmcm Cm c1aC Nm._ Ca_ Cmc + =CCN-PC co zCum AFC C1C+m mm1< NN CNN ozNC o21<C NC CNN CzNC o21<C NC CNN CzNC oz1<C NC CC_ ozNC o21<C NC cecENCC C_C-mC ozNC oz1<C NC C_N CCC <C Nm CCN emm <o Nacommom mcv~+um>ccoo po>wum>vcoo .Cwom ucpoom1M1oFomcc co mwmapmcm mcm compmcmaom on» cam now: mmosuos omgamcmoumEoczo meacvp1mmw11.v w4m wwa no no no no 0" + "a m2-"C]-lndoleacetic acid. specific activity 57.2 mCi/mmole. was obtained from New England Nuclear and used without isotopic dilution: The “C-lAA was purified prior to use by chromatography on a 2 x 22 cm column of Sephadex LII-20 with elution by ethanol-water (1:1). The volatile thiol reagent. dithioethane (Aldrich Chemical Co.. Milwaukee. Wise). was added (10 umole/umole "C-IAA) to protect the "C-lAA from oxidation (cf. 8). The “C-IAA fraction was evaporated to dryness under nitrogen in the presence of 0.56 mole of anthracene (I. T. Baker. 84 14 - 7 -2— A — -I 't Z C Indo.e .. acetyl muo 7.71037. 0 Phillipsburg. New Jersey) per umole of "C-IAA. Anthracene acted to protect the radioactive IAA during the drying step and its presence had no effect on subsequent reactions. Attempts at purification without use of dithioethane and anthracene resulted in almost complete loss ofthe “C-IAA. Purification of "C-lAA prior to use was obligatory. Stoichiometric. rather than catalytic. amounts of sodium methoxide were found to be necessary. possibly because of the difficulty in abstracting a proton from the cyclitol. Sodium methoxide was prepared under dry nitrogen by adding freshly cut sodium metal to anhydrous methanol (11). Methanol was twice redistilled from magnesium activated with iodine (12) onto heat activated Linde 4 A (Supelco. Bellefonte. Pennsylvania) and used immediately. Residual methanol was removed from sodium methoxide in vacuo at 60°C and then by drying for 18 hr at 200°C under high vacuum. This product was stored in vacuo over P20, in a dry nitrogen flushed desiccator. Even small amounts of water resulted in the formation of enough NaOH to hydrolyze the esters as they were made. It was necessary to add sodium methoxide to the reaction as a dry powder since solutions of sodium methoxide in dimethylsulfoxide (DMSO) were ineffective. myo-Inositol (Sigma Chimical Co.. Saint Louis. Missouri) was dried in vacuo in an Abderhalden apparatus at 100°C for 18 hr. DMSO was freshly distilled in vacuo onto Linde 4A. DMSO was stored in sealed 1 ml vials over Linde 4A and the solvent was obtained through a teflon septum as needed. These vials were stored over anhydrous CaSO. and discarded once the septum had been perforated. All glassware was dried at 105°C overnight prior to use. Manipulations involving sodium methoxide were carried out in a glove box in a dry nitrogen atmosphere with P205 present as desiccant. The identity of the radioactive product was established by a number of criteria. First. a parallel synthesis was run on a larger scale using unlabeled IAA and the identity of the products of this synthesis was confirmed as follows. The synthetic products had the same R, values on thin layer chromatograms (E. Merck. Darmstadt. silica gel plates developed in methyl ethyl ketone-ethyl acetate-ethanol-water (3:5:1:l)) as did the naturally occurring mixture of axial and equatorial lAA-inostitols (1). They gave positive reactions with Ehmann's reagent (13) as did the indoleacetyl-myo-inositols isolated from corn seeds. Ammonolysis in 14% NH.OH for 30 min at 45°C yielded two products. one of which co-chromatographed with authentic IAA and the other with indoleacetamide. The products had the characteristic elution volume on a high pressure liquid column (14) and the correct retention times when subjected to gas liquid chromatography (2). Combined gas chromatography/mass spectrometry of any of the four resolvable fully trimethylsilyated derivatives (10) using a LKB 9000 mass spectrometer yielded a molecular ion at m/e = 769 and a fragmentation pattern identical to that previously published for the axial and equatorial esters (10.15). The "C compounds were then compared with the unlabeled compounds. The "C compounds yielded labeled IAA and indoleacetamide upon ammonolysis. Their R, values on thin layer chromatograms and their 327 85 328 J. Nowacki, J.D. Cohen and R.S. Bandurski elution volumes from a high pressure liquid chromatographic column were identical to those of the unlabeled isomeric indoleacetyl-myo-inositols. EXPERIMENTAL After chromatography on Sephadex LII-20. BIZ-“Cl-indoleacetic acid (210 pg. 1.2 pmoles. 68.6 pCi) yielded 175 pg (1 pmole) of purified product. The pooled fractions were evaporated to a small volume on a rotary evaporator and 100 p1 hexane containing 100 pg of anthracene was added. The mixture was dried under nitrogen in a 300 pl microflex tube (Kontes. Vineland. New Jersey). 1.1’-Carbonyldiimidazole (Sigma; 324 pg. 2 pmoles) in 10 pl dry DMSO was added to the dry residue through the septum of the microflex tube and mixed vigorously. Three 1 mm glass beads (B. Braun. Melsungen) were added to aid in dissolving the dry residue. After 30 min of incubation at 25°C. inositol (540 pg. 3 pmoles) in 60 pl DMSO was added with a syringe. The tube was then placed in a nitrogen-flushed dry box and a small amount of sodium methoxide (about 1pmole)was added as a dry powder. The reaction mixture was mixed vigorously for 1-2 min and the reaction stopped by adding 100 pl of ice-cold 2-pr0panol-4 M acetic acid (1:1). Immediately after termination of the reaction the 170 pl of reaction mixture was applied to a 0.9 x 17 cm high pressure column of sulfonated styrene-divinylbenzene copolymer (Beckman PA-28) and separated using 2-propanol-water (1 :1) as the mobile phase (14). Fractions containing significant radioactivity eluted as expected for the isomers of indoleacetyl-myo-inositol and these were pooled. Alcohol was removed in vacuo and the water phase lyophilized onto 100 mg of cellulose powder. The products were eluted from the cellulose with 1 ml ethanol-water (1:1). The yield was calculated from a 5 pl sample of the final solution based on the specific radioactivity of the sample. The yield was 76 pg of "C-indoleacetyl-myo-inositols (13 pCi. 23%) for this synthesis. ACKNOWLEDGEMENTS We thank Dr. W. H. Reusch for advice and Dr. C. C. Sweeley for the use of the mass spectrometer facility (PHS RR-00480). This work was supported by National Science Foundation grant PCM 76-12356 and is journal article 8250 from the Michigan Agricultural Experiment Station. REFERENCES 1. Bandurski R.S..Ueda M.. and Nicholls P. B.-Ann. N.Y. Acad. Scim: 655(1969) 2. Kopcewicz J..Ehmann A..and Bandurski R. S.-Plant Physiol. 51: 846(1974) 3. Hamilton R. H..Bandurski R.S.,and Grigsby B.H.-P1antPhysiol.16: 354(1961) 4. Bandurski R. S. and Schulze A. - Plant Physiol.,SflS): 10 (1977) 86 MST-IndoZe-3—acetyZ-muo-inositol 329 5. Bandurski R. S. - In ”Cyclitols and the Phosphoinositides." Wells. W. W. and Eisenberg. F.. editors. Academic Press. New York. 1977. in press 6. Bandurski R. S.and Schulze A.-PlantPhysiol.fi: 257(1974) 7. Bandurski R. S. and Schulze A.-PlantPhysiol.QQz21l (1977) 8. Felker P.-Anal.Biochem.Zfi192(1976) 9. Staab H. A. -Angew-. Chem. internat. Editi: 351 (1962) 10. Ehmann A.and Bandurski R. S.-Carbohyd. Resggz 1 (1974) ll. Burness D. M. - Organic Syntheses =29: 49 (1959) 12. Riddick .I- A. and Bunger W. B. — “Organic Solvents - Physical Properties and Methods of Purfication." Wiley-lnterscience. New York, 1970 13. Ehmann A.-1.Chromatogr.1_32: 267(1977) 14. Cohen J. D. and Bandurski R. S.-Plant Physiol.5_9[§j: 10(1977) 15. Ehmann A.. Bandurski R. S.. Harten J.. and Sweeley C. C. - "Mass Spectrometry of Indoles and Trimethylsilyl-indole Derivitives." Michigan State University. East Lansing. Michigan. 1975 87 EXPERIMENTAL IV AUTOMATED ANALYSIS OF INDOLYLIC COMPOUNDS IN PLANT EXTRACTS Jerry D. Cohen Department of Botany and Plant Pathology Michigan State University East Lansing, MI 48824 Running Title: Indole analyzer Received: 88 Abstract An automated procedure for analysis of indolylic compounds has been developed utilizing a chromogenic reagent yielding stable chromogens with EEm = 24,000. Coupling this detection procedure to an adsorption chromatographic system of sulfonated polystyrene divinylbenzene, such as used in automated amino acid analysis, per— mitted separation, detection and assay of indoles in crude extracts of kernels of Zea mays. Analysis of the major neutral and acidic indoles in crude extracts was accomplished in less than 3 hours, where previous methods required several days. Adaptability of this method to other tissues and column chromatographic systems is des- cribed. These methods describe the first versatile indole analyzer useable with crude plant extracts. The analyzer should have signif- icant applications in assays of indoles in both plant and animal extracts. Introduction Derivatives of the indole nucleus are important in the nutri- tional and hormonal biochemistry of all higher organisms (cf. Bandurski, l978; Lovenberg and Engelman, 1971) and automated analysis of indolylic compounds would be important in assays of the plant growth hormone, indole—3-acetic acid (IAA); in nutritional studies of tryptophan concentration; and in studies of biogenic amines in body fluids and in plants. Automated analysis has previously been difficult owing to the lack of a stable-colored indole derivative with sufficiently rapid formation for automation. In addition, 89 previous chromatographic procedures which were capable of selectively concentrating and separating indole compounds in crude plant extracts (Ueda and Bandurski, l969; Ehmann and Bandurski, l972; Magnus et al., l978) were very slow and this protracted elution restricted attempts at automation. In plant tissues most of the IAA exists not as the free acid, but bonded via an ester or amide linkage to other compounds (Bandurski and Schulze, 1977). In Zea mays about half of the IAA is in the form of low molecular weight esters conjugated with inositol or inositol-glycosides (Bandurski et al., l969). These conjugates play important roles in the hormonal physiology of the young seedling (Bandurski, l978) and new methods for analysis of these compounds are needed. This work describes a system for the automated analysis of indoles using a modification of the Ehmann (1977) indole reagent. In addition a new column for the analysis of neutral and acidic plant indoles by absorption chromatography is described. The column method offers a purification superior to previous low pressure aqueous column methods and provides a 25-fold reduction in analysis time. Together, these methods comprise the first automated indole analyzer suitable for use with plant extracts. An abstract of this work has previously appeared (Cohen and Bandurski, l977). 90 Materials and Methods Reagents Indole-3-acetic acid was from Sigma, indole-3-acetyl-L- aspartic acid was a gift from R. Hangarter and N. E. Good (Michigan State University) and the isomeric indole-3—acetyl-myo-inositols were synthesized by the method of Nowacki et al. (1978). The inositol derivatives were purified as previously described (Cohen and Bandurski, l978). Ehmann's reagent was identical to that reported (Ehmann, 1977), a 1:3 mixture of Van Urk (l929) and Salkowski (Tang and Bonner, l947) reagent freshly mixed from stocks of each. The Van Urk reagent was prepared from p-dimethylaminobenzaldehyde (Aldrich) decolorized by refluxing 200 g with l g norite in 500 ml absolute ethanol and recrystallized from ethanol-water to yield large off-white leaflets which were dried in partial vacuum over P205 for one week. Reagent p-dimethylaminobenzaldehyde (Sigma) is suitable, although sensitivity is reduced by 30%. The Van Urk reagent was stable for about one month and was prepared by dissolv- ing 1 gm p-dimethylaminobenzaldehyde in 50 ml of reagent grade HCl (specific gravity = l.lB) and adding 50 ml of absolute ethanol which had been redistilled after mixing with 5 g/l of norite for l8 hours. Salkowski reagent was prepared by dissolving 2.03 g FeCl3o6H20 (Baker) in 500 ml of glass distilled H20 and adding 300 ml reagent grade H2304 (specific gravity = 1.84). Water used in reagents and for dilution in the analyzer was distilled, deionized and redistilled so that the conductance was below l umho. 91 Plant Material Corn kernels (Zea mays L. cv Stowell's Evergreen, Vaughan's Seed Co., Ovid, Mich.) were ground in a mechanical hammermill to about 20-50 mesh and 100 gram aliquots were extracted with 3 por- tions of 300 ml of 80% aqueous acetone (v/v) during l6 hours. The combined extracts were reduced to 150 ml and stored l6 hours at 4 C to permit oligosaccharide retrogradation and precipitation. Filtration through Whatman l and 42 paper yielded a clear yellow solution which was then reduced in volume to 50 ml and applied to a 4.5 cm x 4 cm bed of Na+ form Dowex 50x2-4oo (Sigma). The Dowex was eluted by successive washing with l00 ml aliquots of 50% 2-propanol/water (v/v) using vacuum to aid filtration. The first 200 ml were discarded since they contained mainly saccharides and little or no indole compounds detectable by tlc (see below). Puri- fication by Dowex 50 was essential to protect the analyzer resin from irreversible binding of damaging compounds and to remove sugars so the sample could be reduced to l200 ul, the maximum column load- ing volume. Thin Layer Chromatography Thin layer chromatography for the separation of indole derivatives was on silica-gel 60 plates (56l0; E. Merck, Darmstadt) using ethyl acetate, methyl ethyl ketone, ethanol, water (5:3:lzl) as solvent (Labarca et al., l965). Indoles were visualized by spraying with the Ehmann reagent and with color development at 100 C for five minutes. Washing the developed plate in distilled 92 water removed acidic residues and permitted storage of the plates with spot color retention (Ehmann, l977). Column Regeneration PA—28 resin was regenerated by treatment with 70% H2504 for 5 hours at 70 C. The acid-resin was diluted with 4 volumes of water and washed with water in a buchner funnel until neutral. Next the resin was mixed with 0.l% ethylenediaminetetraacetic acid (EDTA) disodium salt (Sigma) in l N NaOH and again washed with water until neutral. This procedure was repeated with 2 N HCl and finally with l N NaOH. The resin was then washed with 50% 2-propanol/water (v/v) until the eluent was colorless. The Partisil ODS column was eluted with several bed volumes of absolute ethanol for regeneration and then stored with absolute ethanol to avoid dissolution of the silica gel support matrix. At least 5 bed volumes of solvent was passed through the column prior to a chromatographic separation. Column Chromatography Chromatographic separations were on a 0.9 x l7 cm column of spherical sulfonated polystyrene divinylbenzene resin designed for amino acid analysis (Beckman PA-28). A pressure of 10 atm was provided by a Beckman Accuflo pump yielding a flow rate of 0.3 ml/ min with 50% 2-propanol/water (v/v) as the eluting solvent. Sample application was with a teflon Altex isolated loop type injection valve (Figure 1). Plumbing was with 1.5 mm ID teflon tubing except fbr the injection valve which was internally plumbed with 0.8 mm ID 93 tubing. Injection valve loops of 20 pl, 500 pl, and l200 pl were used. A second column used in these investigations was a 4 mm x 25 cm l0 p Partisil ODS column (Whatman) or a "micro-bondapak Cl8" column (Waters) operated at 150 atm by a Milton Roy 396 minipump equipped with a Rheodyne 7l20 injection valve and a pulse damping system. Elution was isocratic with 20% ethanol/water (v/v) contain- ing l% acetic acid. Samples eluting from these columns were "split" with a nylon ”T” union (Value Plastics, Inc., Loveland, Colo.) on the 0.8 mm ID teflon column end tubing so that approximately 20% entered the autoanalyzer and 80% was collected in l ml volumes on a Gilson FC80K microfractionator. During initial column development the auto- analyzer was not available so the standard samples were simply col- lected and analyzed by measuring ultraviolet (U.V.) adsorption at. 282 nm on a Gilford 240 spectrometer using l ml far U.V. quartz cuvettes. Autoanalyzer A diagram of the analyzer is shown in Figure 2. For use with the PA-28 column the sampling rate was 67 pl/min through 0.25 mm ID tubing. Other tubing sizes were adjusted such that the ratio of sample:air:reagent:water was l:4:l6:42.5. Critical values for proper analysis were: (1) The air bubble must be l/2 to l/5 the volume of reagent. Too many bubbles results in poor mixing and increases loss during "debubbling." (2) Sample volume must not be 94 greater than 25% of reagent volume, with less than 10% preferred. (3) Water should be about twice the reagent volume. Less water results in excessive background absorbance and larger amounts of water unnecessarily dilutes the color. The only modification neces- sary for use with the Partisil ODS column was an increase in sample size to 270 pl/min because of the higher column flow rates. The colorimeter used during initial development was the standard fixed wavelength (570 nm) Technicon colorimeter. This was found to be unsatisfactory, however, since a loss of sensitivity resulted from the non-optimal wavelength and because with this colorimeter a stable baseline was difficult to obtain. Superior performance was obtained with a 3 channel colorimeter (M.E.R. Chromatographic, Mountain View, Calif. 94040, model 1020 with a model 1050 long-path channel add-on) operated at 5.0 volts by a 0-7.5 volt 10A regulated power supply (Model IP-2730, Heath Co., Benton Harbor, Mich. 49022). The colorimeter was equipped with 6.6 mm and 20 mm flowcells with 610 nm filters as well as a 6.6 mm flowcell at 800 nm with a near infrared photocell (M.E.R. Chroma- tographic, Model 25). The indole product from Ehmann's reagent has no absorbance at 800 nm, so this channel provided a measure- ment for turbidit --the major interference found in our work with this reagent. This colorimeter proved to be extremely stable and, in cost, represented a considerable savings over even the single channel Technicon unit. Output was to a Honeywell Electronik 15, 3 channel 5 mv multipoint recorder using 1000 ohm 10 turn helipots (M.E.R. Chromatographic, Model H-1000) for zero control. 95 Results and Discussion Chromatography on PA-28 Resin Beckman PA-28 resin is a spherical sulfonated polystyrene- divinylbenzene copolymer which, in 50% 2-propanol/water (v/v), has an average diameter of 23.2 i 5 pm as measured with a microscope equipped with an ocular micrometer. The indoles used in this study were acidic or neutral and do not bind ionically to the strongly acidic resin. Adsorption of these compounds is attributable to non-electrostatic attraction due to hydrophobic or Van der Walls- London forces (Niederwieser, 1971). These forces also influence binding of amino acids (Neiderwieser, 1975), but the binding forces for indoles on polystyrene are much stronger than those for the amino acids (Niederwieser and Giliberti, 1971). Tryptophan exhibits the highest affinity for polystyrene of any of the amino acids (Niederwieser, 1971), however its maximum absorption coefficient is only one tenth as high as that for indole or indole-3-acetic acid (Niederwieser and Giliberti, 1971). The retention of IAA-myo- inositol and the glycosides of IAA-myo-inositol may be attributable to coplaner structures and also because myo-inositol and simple sugars, as well as the indolylic moiety, are retained by the column. Free rotation should allow both ring systems to interact with the resin. Separation of these compounds on similar resins using con- ventional liquid chromatography was described by Ehmann and Bandurski (1972). The elution profile of a mixture of IAA and synthetic mixed isomeric IAA-myo—inositols is shown in Figure 3. The first peak is 96 IAA and the slower eluting double peaks are the IAA-myo-inositols. The inositol esters coelute with only some separation of the isomeric forms. These compounds exhibit acyl migration during preparation so that purification and concentration as a group is advantageous. Based on thin layer migration (Ehmann and Bandurski, 1972) it is possible to show that the first peak is a mixture of axial and one or more equatorial isomers and that the later shoulder is devoid of the axial isomer. As can be seen (Figure 3) flow rates faster than 14 ml/hr resulted in zone spreading and a decrease in retention time. Further reduction of flow rates to 8 ml/hr did not improve separa- tion. Increasing column temperature from 25 C to 50 C markedly changes the elution profile (Figure 4). The retention of IAA is increased slightly and the IAA-inositols elute earlier. Although slight tailing is still observed, the characteristic double peak is lost and the compounds elute in a smaller volume. This change in elution pattern could be advantageous for some separations where multiple passages through the column may be required. Recovery of indoles from the PA-28 column is a function of the amount applied. Recovery averages 70% or better with samples of IAA of 3 pg or above whereas with samples of 1 pg the recovery drops to 30%. Addition of thioglycerol to the solvent and purging the solvent with N2 had no effect on the recovery. The resins were EDTA treated so it is unlikely that metal contaminants accounted for this reduced yield. The loading capacity of the column is high and samples of synthetic IAA-myo-inositols in excess of 25 mg have 97 been purified on this column. The useable range for this column is, therefore, 1 to 25,000 pg. Chromatography of Indoles on a C18" Reverse Phase Column The purification of simple indoles by HPLC on a C18-reverse phase column has been previously reported for samples of animal (Graffeo and Karger, 1976; Balandrin et al., 1978; Anderson and Purdy, 1979) and plant origin (Sweetser and Swartzfager, 1978; Brenner, personal communication) using relatively nonspecific methods of detection. Graffeo and Karger (1976) described a system for indole analysis in urine using fluorometric detection which has also been applied to samples from cerebrospinal fluid, brain, and plasma (Anderson and Purdy, 1979). Other authors have detected indoles using U.V. and electrochemical detection (Sweetser and Swartzfager, 1978) and while these methods are very sensitive, they are not applicable to crude plant samples because of the presence of vast excesses of flourescent and U.V. absorbing phenolic acids. A study of recovery from reverse phase columns has been 14C labeled IAA conducted by A. Schulze of this laboratory. Using she found that recovery averages about 70% with submicrogram samples. Recovery falls to about 50% when plant samples prepurified on DEAE- Sephadex are analyzed. Interestingly, as the column ages the recovery of standards remains high while the recovery of IAA from the plant samples drops percipitously to about 25%. These low recoveries are probably due to the 50% of the available surface area of the support matrix that is not occupied by the C18 groups. 98 We believe that minute amounts of indoles are subject to oxidative attack by many compounds when absorbed to acidic supports. Autoanalyzer Automated assay is based on the Ehmann (1977) reagent, which is a sensitive and specific chromogenic reagent for indoles. Many indoles substituted on C(3) give products with a spectrum such as shown in Figure 5 for IAA. The extinction coefficient for this product at 615 nm is 24,000, or four times the extinction for indoles at 282 nm. Spectra and extinctions identical to that of IAA are obtained with IAA-myo-inositol and several amino acid amide conju- gates of IAA (Cohen and Bandurski, 1978). The spectrum for the pro- duct of Ehmann's reagent with tryptophan has an optimum at 590 nm and an extinction of 17,000. However, the spectrum is broad and readings at 570 or 610 nm are within 8% of the absorbance at maxi- mum. Numerous other indoles react, as described by Ehmann (1977), and could be analyzed using this reagent. This reagent is superior to the Salkowski reagent of Tang and Bonner (1947), since it is more selective for indoles and because the chromophore color develops rapidly and is stable. Compounds which interfere with the reagent are (1) those compounds which, in acid, yield insoluble products which impart turbidity, (2) large excesses of phenolic compounds (Ehmann, 1977), and (3) reducing agents. The chromophore formed by reaction of the Ehmann reagent with IAA obeys Beer's law and is a linear function of amount of IAA over the interval 0.5 to 10 pg. These experiments were done by 99 direct injection of IAA into the analyzer inlet stream (Figure 6) using a 6.6 mm flow cell. With a 20 mm flow cell, samples as small as 100 ng are measurable with a 10:1 signal to noise ratio and fall on the extrapolated calibration line. Zone spreading within the analyzer is small as is indicated by plate number calculations. With 2 pg sample of IAA assayed by U.V. absorbancy at 280 nm, the plate number for the reverse phase column was 1120 (see Saunders, 1975 for calculations); whereas when 10 pg was injected onto the column and 20% of the eluent autoanalyzed, the plate number was 900. Since some of this loss of efficiency is due to increased column loading, zone spreading due to the autoanalyzer must be less than 20% of peak width. The utility of the autoanalyzer is detecting indole com- pounds in crude samples is shown in Figure 7 and 8. A thin layer chromatogram of fractions eluted from the PA-28 column after appli- cation of a crude corn extract is shown in Figure 7. The plate was sprayed with Ehmann's reagent (Ehmann, 1977) after solvent develop- ment. A large amount of charable material was eluted in early fractions. ‘These compounds lead to turbidity and a general rise in baseline is seen between 30 and 80 min in Figure 8. Some phenolic compounds yielding a pink color and some unidentified faint blue colored spots result in early peaks. The column retains IAA longer with crude extracts than when standards are chromato- graphed, probably because acidic compounds in the extract protonate ‘the IAA and the undissociated IAA is more strongly adsorbed to the resin matrix (Niederwieser and Giliberti, 1971; Ehmann and 100 Bandurski, 1972). IAA-myo-inositols elute in a sharper peak from crude plant extract than from standards (compare Figures 7 and 8 with Figures 3 and 4), and this could be owing to lipodial plant 'compounds affecting entry of indoles into the stationary phase. The indole analyzer is also useful for the detection of indole-3-acetyl-L-aspartic acid, which elutes slightly before the IAA-myo-inositols with a 14 ml elution volume. IAA-aspartic acid is another important IAA conjugate which has been identified after feeding Pisum sativum seedlings large amounts of exogenous IAA (Andreae and Good, 1955), however its presence as a natural product has not been demonstrated by chemically rigorous procedures. Coupling the PA-28 column to the Ehmann assay provides the first automated system for the analysis of indoles in crude plant extracts. In this laboratory these methods have proved useful for the preparative scale purification of IAA esters from corn, the rapid purification of synthetic IAA—myo-inositols (both high spe- cific activity 14C labeled (Nowacki et al., 1978) and preparative scale purification (Cohen and Bandurski, 1978)), the identification of IAA-myo-inositol formed by enzymatic synthesis (Michalczuk and Bandurski, 1979), and the first isolation of IAA-myo-inositol from rice (Hall and Bandurski, 1979). The analyzer is not limited to use with the PA-28 column and can easily be adapted to other columns, such as the C18 column also used in this study. This versatility makes the analyzer useful for the analysis of at least 80 different indoles which react with the reagent (Ehmann, 1977). This method offers greater sensitivity for tryptophan assays than 101 a previously reported method (Amaya-F et al., 1977) and methods for the rapid separation of numerous tryptamine (Balandrin et al., 1978; Villanueva and Adlakha, 1978) and indole (Graffeo and Karger, 1976; During, 1977; Sweetser and Swartzfager, 1978; Anderson and Purdy, 1979) derivatives should be easily adapted to the analyzer. 102 Literature Cited , Young, C. T., Chichester, C. 0., Agric. Food Chem. Amaya-F, J. 25,139 (1977). Anderson, 6. M., Purdy, u. C., Anal. Chem. 5,283 (1979). Andreae, W. A., Good, N. E., P1ant Physiol. 2Q,380 (1955). Balandrin, M. F., Kinghorn, A. D., Smolenski, S. J., Dobberstein, R. H., J. Chromatogr. 157,365 (1978). Bandurski, R. S., "Cyclitols and the phosphoinositides," Eisenberg, F., Hells, w. H., Eds., Academic Press, New York, N.Y., 1978, pp. 35+54. Bandurski, R. S., Schulze, A., Plant Physiol. 69,211 (1977). Bandurski, R. S., Ueda, M., Nicholls, P. B., Ann. N.Y. Acad. Sci. fifiss (1969). Cohen, J. D., Bandurski, R. 5., Plant Physiol. 61(3),10 (1977). Cohen, J. D., Bandurski, R. S., Planta 139,203 (1978). During, H., Experientia 22,1666 (1977). Ehmann, A., J. Chromatogr. 132,267 (1977). Ehmann, A., Bandurski, R. S., J. Chromatogr. 22,61 (1972). Graffeo, A. P., Karger, B. L., Clinical Chem. 22,184 (1976). Hall, P. J., Bandurski, R. 5., Plant Physiol. 62(3),50 (1979). Labarca, C., Nicholls, P. B., Bandurski, R. 5., Biochem. Bigphys. Res. Comm. 29,641 (1965). Lovenberg, w., Engelman, K., "Methods of biochemical analysis- analysis of biogenic amines and their related enzymes," Glick, D., Ed., Wiley, New York, N.Y., 1971, pp. 1-34. Magnus, V., Iskric, S., Kveder, S., Croatica Chemica Acta 51,177 (1978 . ' Michalczuk, L., Bandurski, R. 3., Plant Physiol. 62(8),50 (1979). Niederwieser, A., J. Chromatogr. 61,81 (1971). 103 Niederwieser, A., "Chromatography, a laboratory handbook of chroma- tographic and electrophoretic methods," Heftmann, E., Ed., Van Nostrand Reinhold Company, New York, N.Y., 3rd ed., 1975. pp. 393-465. Niederwieser, A., Giliberti, P., J. Chromatogr. 61,95 (1971). Nowacki, J., Cohen, J. D., Bandurski, R. S., J. Label. Comp. Radio. 16,325 (1978). Saunders, D. L., "Chromatography, a laboratory handbook of chroma- tographic and electrophoretic methods," Heftmann, E., Ed., Van Nostrand Reinhold Company, New York, N.Y., 3rd ed., 1975, pp. 77-109. Sweetser, P. B., Swartzfager, D. 0., Plant Physiol. 61,254 (1978). Tang, Y. H., Bonner, J., Arch. Biochem. 16,11 (1947). Ueda, M., Bandurski, R. 8., Plant Physiol. 44,1175 (1969). Van Urk, H. H., Pharm. Neekbl. 66,473 (1929). Villanueva, V. R., Adlakha, R. C., Anal. Biochem. 61,264 (1978). Fig. 1 Fig. 2 Fig. 3 104 Legends for Figures Diagramatic representation of the PA—28 chromatographic system. Splitting the effluent after the column permits 20% of the sample to be analyzed and the remainder to be collected hithe fraction collector (F.C.). Automated analyzer for the determination of indoles with the Ehmann reagent. Reagent and sample are drawn into a mixing manifold where an air bubble is introduced. The solution is mixed and then heated at 65 C for 5 min by passing through 3 meters of 1.35 mm ID teflon tubing in a water bath. After cooling in a condensor, water is added and the solution is again mixed. The air bubble is removed prior to measuring the absorbance at 610 nm. Pump tubing is tygon except for the reagent line which is "acidflex." The glass coils and manifolds are from Technicon, all other tubing is teflon. The effect of the rate of solvent flow on the elution profile of a mixture of IAA and mixed isomeric IAA-myo- inositols from the PA-28 column as measured by U.V. absorbance. Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 105 The effect of column temperature on the elution profile of a mixture of IAA and mixed isomeric IAA-mya-inositols from the PA-28 column. Column temperature was controlled by circulating water through a water jacket on the column. Absorbancy of the Ehmann product of IAA as a function of wavelength using a Cary 15 spectrophotometer. Calibration of the autoanalyzer by injection into the sample stream and using 6.6 mm flow cells. Units refer to area under the peak. Thin layer chromatogram of fractions from the PA-28 column of a crude extract of Zea mays kernels. Details are as indicated in the text and areas indicated by letters correspond to regions in Figure 8. The compounds in later fractions are: Rf = 0.90, IAA; Rf = 0.39 and 0.35, IAA-myo-inositol; Rf = 0.23, 0.18, and 0.13, IAA-myo-inositolglycosides. Recording of the elution profile as visualized with the analyzer for a crude extract from Zea mays kernels. Regions indicated by letters correspond to those shown in Figure 7. > v 6:32 8 :dm .u .u o» zSDJOU 106 hzm>gom DU] mesa \J \ >— g w>4<> zomewzH 335 5.38% 107 Ihozwm xmz QED; ozfizofihmomomm 9 m~< H2uwo>> 00m CON 000 com 00V _ p . _ p . _ . .oo 1 .md T o? .o._ aouquosqv 111 1010 do 2.0 ”g. 112 —D 1.0 - ITTI|IIII| ' 3 8 ’u f 4‘. {'1' 113 cap Cm, 2232: o... oo J" r "“1"" ...\..<. 5}?! .- _..0 WV 0 No 114 EXPERIMENTAL V DOUBLE STANDARD ISOTOPE DILUTION ASSAY I. QUANTITATIVE ASSAY 0F INDOLE-3-ACETIC ACID Jerry D. Cohen and Robert S. Bandurski Department of Botany and Plant Pathology Michigan State University East Lansing, MI 48824 Running Title: Double standard assay Received: 115 Abstract Isotope dilution analysis for the quantitation of labile compounds has been limited by the amount of sample necessary to redetermine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determi- nation of specific activity by gas chromatography. It requires the availability of the radiolabeled compound to be analyzed and also requires a chemically-related radiolabeled compound. The method is illustrated by assaying indole-3-acetic acid in plant extracts using 14C-indole-3-acetic acid and adding 14 C-indole-3-butyric acid at the final stage of analysis prior to gas chromatography. Used with a nitrogen specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of 14C-[2-ring]- indole-3-butyric acid is also described. 116 Double Standard Isotope Dilution Assay I. Quantitative Assay of Indole-3-acetic Acid Isotope dilution analysis, described in 1940 by Rittenberg and Foster (1), can be used with stable or radioactive isotopes and is the method of choice for quantitative analysis of labile natural products. Use of this method requires knowledge of the initial specific activity of the applied labeled compound and determination of the final specific activity after dilution of the isotope by the endogenous compound being assayed. Ability to accurately assay radioactivity rarely limits the sensitivity of the method since high specific activity compounds are generally available. It is the accuracy with which the amount of compound recovered can be assayed by chemical means that limits sensitivity. In this work we describe a technique that quantifies gas-liquid chromatography using highly sensitive detectors and thus brings the sensitivity of the chemical determination to approximately the same level as that of the radio— isotope assay. The method, which we have named “double standard isotope dilution assay,” should have general applicability. It requires the availability of the isotopically labeled compound to be assayed and that a chemically-related, isotopically-labeled com- pound also be available. This second, chemically-similar labeled compound permits determination of the specific activity of the com- pound being assayed using gas—liquid chromatography. We illustrate the method by assay of indole—3—acetic acid (IAA)——a labile (cf. 2) growth hormone present in plant tissues in minute amounts (3). Nano- gram sensitivity is attained by use of the double standard method and the nitrogen specific thermionic detector for gas-liquid chromatography. 117 Materials and Methods The Double Standard The assay procedure involves purification of endogenous and added 14 C-labeled IAA to the step just prior to gas-liquid chroma- tography. Then, 14C-labeled indole-3—butyric acid (IBA) is added to the IAA in approximately equivalent amounts. The mixture is then derivatized and analyzed with a suitable gas chromatography detector. Knowing the peak area of IBA and the amount of IBA added, the amount of IAA in the sample at the time of derivatization may be calculated by the relationship: %%%—%%Efi%§€§§%-X amount of IBA added = amount of IAA p in sample at time of derivatization Similarly, if a replicate injection is made and the radio— activity in the IAA and IBA collected then the radioactivity in IAA at the time of derivatization may be calculated: IAA pCi collected) . _ . . . x IBA (pCi added) — pCi of IAA in sample at IBA “61 collected time of derivatization Then knowing the amount of IAA in the sample and the radioactivity: IAA (pCi in sample) IAA (amount in sample ) = specific activity of IAA This is the diluted specific activity of the 14C-IAA originally added to the plant extract and thus the isotope dilution equation of Rittenberg and Foster (1) may be applied: where Y = amount of compound in sample, Ci = the initial specific activity of the applied labeled compound, Cf = the final specific 14 activity, as determined above and x = the amount of C-IAA initially added. 140- In practice it is convenient to add 2—3 times as much IBA as IAA anticipated in the unknown. Similarly we adjust the spe- cific activity of the IBA to be approximately equal to that of the IAA as reisolated. These precautions, while not essential, enhance the accuracy of the method. Likewise, the removal of an aliquot from the sample prior to the addition of the 14 C-IBA provides a sample for use in checking for compounds which might coemerge with the IBA peak during gas-liquid chromatography. Determination of Specific Activity Determination of peak areas and radioactivity can be accom- plished in two separate injections or in one injection with an effluent splitter. If the single injection system is used it is advisable to use an annular splitter at the collection port since this splitter maintains a constant split ratio even with changing mass load (see 4 for details of splitter and sample collection). If two injections are used then, during one injection, the flame is extinguished and glass tubes placed over the jet for collection at the correct retention times. We have found this method of col— lection to be better than 98% efficient. After collection, the 119 samples are rinsed into vials containing ACS scintillation fluid (Amersham) and counted on a Packard 3003 liquid scintillation counter. From the other injection, or from the recording of the split injection, relative peak areas can be determined. From these two pieces of experimental data, the relative peak areas and radio- activity, and thus the specific activity of the IAA can be deter- mined as described above. Preparation of 14C-indole- 3-butyric acid The synthesis of 14C—[2—ring]-indole-3-butyric acid (Figure 1) was by a microscale modification of the industrial process of H. E. Fritz (5). Indole (Eastman) was recrystallized from water- ethanol and 12 mg of the dry crystalline leaflets were added to a 1 ml size (4 ml capacity) freeze—drying tear bulb (A. H. Thomas 5136-610) fitted with a condenser collar. To this was added 10 p1 of 1,2-ethanedithiol (Aldrich) (4,6) and 50 pCi 14C—[ring 2]-indole (50 mCi/mmole, ICN Pharmaceuticals). The 1,2-ethanedithiol destroys 14c-indoie to be dried peroxides and free radicals permitting the without decomposition (6). The hexane solvent and 1,2-ethanedithiol (stench) were removed under a stream of dry N2. To the dry residue was added 0.75 g of freshly broken NaOH pellets and 1.5 ml of y- butyrolactone (Aldrich). The vessel was then placed in a thermo- stated sand bath and the temperature of the sand was brought to 220 C at the rate of 2 C/min. A slow reflux was maintained for an additional 23 hours and the reaction terminated by the addition of water. The solidified reaction mixture was dissolved in the water 120 by warming and the vessel rinsed repeatedly so the final volume was 50 ml. The water solution was shaken twice with an equal volume of chloroform and the chloroform discarded. The water phase was adjusted to pH 2.5 with 5 N HCl and this extracted twice with chloro- form. The combined chloroform extracts were dried over anhydrous sodium sulfate, filtered and brought to near dryness in vacuo at 45 C. The residue was dissolved in 2 m1 of 50% 2-propanol/water (v/v) and applied to a 3 x 30 cm Sephadex LH-20 column equilibrated with 50% 2-propanol/water (v/v). IBA was eluted with 50% 2-propanol/ water (v/v) at 244 to 276 ml. Yield, based on indole or radio- activity, was 3 %. Purity of the product was checked by gas-liquid chromatography of the trimethylsilyated derivative on a 4 ft., 2 mm ID column of 5% SP-2401 on 100/120 Supelcon Aw-DMCS (Supelco) equipped with an annular effluent splitter. Collection of the radio- activity showed that less than 0.4% of the radioactivity emerged prior to the IBA peak, 93.3% was contained in the peak at the reten- tion time of authentic bis-TMS—IBA and the remainder of the counts, 6.3%, were in a small shoulder at the retention time expected for the mono-TMS—IBA. Product identity was established using several criteria. First, the putative IBA behaved exactly as the authentic IBA in its chromatographic behavior on Sephadex LH—20, and during silica gel 60 thin layer chromatography (chloroform, methanol, water; 85:14:l and detection with Ehmann's reagent (7)). Gas-liquid chromatography of the trimethylsilyl derivative showed a retention time of 10.4 min 121 for both authentic and putative IBA. The Cary 15 (Applied Physics) U.V. spectrum of the product showed peaks at 282 and 222 and shoulders at 274 and 289 (Figure 2) as for authentic IBA. The 70 eV mass spectra (combined gas-liquid chromatography/mass spectrometry on an LKB 9000) of the methyl ester showed a molecular ion at 217 and base peak at 130 (Figure 3) as for authentic IBA. Gas Chromatography Gas-liquid chromatography was on a Hewlett-Packard model 402 equipped with a flame ionization detector (FID) with an annular effluent splitter and a Tracor 702 nitrogen-phosphorus specific thermionic detector (N2 ). Nitrogen and helium were used as carrier gas for the F10 and N21 detectors, respectively. The columns used during these investigations were (1) 4 ft x 2 mm ID glass column packed with 5% SP—2401 on 100/120 Supelcon Aw-DMCS (Supelco) for use with TMS derivatized samples, (2) 4 ft x 2 mm ID glass column packed with 3% 0V-l7 on 100/120 Gas Chrom 0 (Applied Science) for methyl esters, and (3) a 10 ft x 2 mm ID glass column packed with 3% 0V-225 on 80/100 Chromosorb w-HP for gas chromatography-mass spectrometry analysis of plant samples on a Hewlett Packard 5985a instrument. Several methods were utilized for the formation of volatile derivatives for gas-liquid chromatography. For the addition of trimethylsilyl groups the sample was dried under a nitrogen stream at 50 C, closed with a rubber septum and resuspended in pyridine (approx. 0.5 ullug IAA). An equal volume of N,0-bis-(trimethylsi1yl)- trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) 122 (Pierce) was added and the mixture reacted for 30 min at 45 C. For the formation of the methyl ester of IAA the most satisfactory method was with the use of diazomethane as described by Schlenk and Gellerman (8) for fatty acids. Use of 10% methanol in the reaction solution and the use of freshly redistilled, peroxide free ether was found to be obligatory if high yields were to be obtained. Finally, the N—hepta— fluorobutyryl—indole-3-acetic acid methyl ester was formed by react- ing the IAA-methyl ester with 10 p1 heptafluorobutyric anhydride (Pierce) and 2 p1 of a solution of 0.1% dimethylaminopyridine in tetrahydrofuran. The reaction mixture was heated for 5 min at 100 C and blown dry with N2. This N-acylation procedure was carried out in a nitrogen flushed dry box (9) with P205 present as a desiccant. The use of the dimethylaminopyridine acylation catalyst (10) sub- stantially improved yields when working at the submicrogram level. Plant Material Corn kernels (Zea mays L. cv. Stowell's Evergreen; Vaughan's Seed Co., Ovid, Michigan) were grown in the dark in moist paper towels for 5 days at 25 C as previously described (11). Shoots were removed from the seedlings and samples of 50 9 ground in 100 ml 80% aqueous acetone (v/v) in a Waring blender. Immediately after grind- -14C)-indole-3—acetic acid (New England Nuclear, ing, 0.039 pCi of (2 57.2 mCi/mmole) was added to the homogenate. After gravity filtra- tion through Whatman #1 filter paper the residue was made to 1 N with NaOH and held for 1 hr at 25 C. The filtrate was acidified with H2304 to pH 2.5 and extracted with 50 ml chloroform three 123 times. Chloroform phases were pooled, dried over anhydrous sodium sulfate and reduced to dryness in vacuo at 45 C. The residue was dissolved in 250 pl of 50% ethanol/water (v/v) and placed on a 0.7 x 12.5 cm DEAE-Sephadex-acetate A-25 (Sigma) column equilibrated with 50% ethanol/water (v/v). After washing with 24 ml of solvent, the IAA was eluted with a linear gradient of 0-2.5% acetic acid in 50% ethanol/water (v/v) starting with 100 ml of 50% ethanol/water (v/v) in the mixing flask and 100 ml of 50% ethanol/water containing 5% acetic acid (v/v/v) in the reservoir flask. IAA was eluted between 40 to 46 m1 after beginning the gradient. 4 The IAA containing peak was pooled, dried, dissolved in 0.5 ml water and applied to a l x 15 cm column of bovine serum albumin coupled to Sepharose. This column was prepared by reacting 30 ml (settled volume) of Sepharose-4B with 3.5 g of cyanogen bromide at pH 11.5 and then reacting the activated Sepharose with 750 mg of the serum albumin (12). The column was eluted with 60 ml 0.01M potassium phosphate, pH 5.5, then with buffer saturated with benzene. A more complete report of the preparation and use of this column will be the subject of a future report from this laboratory. IAA eluted after approximately 20 ml of the benzene buffer. An additional method of purification useful in place of, or in conjunction with, the BSA-Sepharose column in ClB-reverse phase high pressure liquid chromatography on a 25 cm x 4 mm Nhatman 10 p Partisil ODS column with isocratic elution with 20% aqueous ethanol containing 1% acetic acid. 124 Results and Discussion The use of indole analogs as internal standards for quanti— tative determination of IAA have been previously described. For example, 5—methyl-IAA (13,14) IBA (15) and indole-3-propionic acid (16) have been used to correct for losses of IAA during isolation. These methods are unsatisfactory since isolation of IAA is a multi- step procedure and these compounds are fractionally lost at several steps during purification (17). To alleviate this difficulty, iso- topically labeled IAA is employed as an internal standard and 3H (18), ‘4 (22)) labels have all proved useful. Deuterated internal standards c (3,19) and 2H (side chain, 2d (20,21) or ring, 4d and 5d however require routine access to a mass spectrometer. The method here described increases the sensitivity of the radioisotope method to about the sensitivity obtained with GC-MS-SIM (selected ion minotoring)--that is to 1-10 ng for reproducible quan- titation of IAA (20, 21 and A. Schulze, unpublished). GC-MS-SIM is, of course, of variable sensitivity depending on machine design and 14C-IBA eliminates the four major source cleanliness. The use of the sources of error in quantitative GC: (1) The volume used for assay of microliter samples of volatile solvents is difficult to control and evaporative losses of microliter samples occur even from "sealed” vials. (2) Precise control of injection volumes in the microliter range is difficult and although an attempt should be made to control injection volume for this method (23,24), errors resulting from even large changes in injection volume are small. (3) Derivatization of sample is always variable and incomplete and the extent of 125 derivatization with standards may differ from that with natural pro- duct extracts. (4) Finally, detector response on gas chromatographs Tchange with each run. This is true for FID (4) and N2T (25) detec- 14C-IBA internal standard tors. For these reasons, the use of a added at the last step of the assay is superior to previously des- cribed methods. The synthesis of 14C-IBA was accomplished by treatment of indolyl sodium with y-butyrolactone (5) yielding the C(3) substi- tuted indole acid. Probably two different reactions occur. First, during the slow initial hearing period, alkylation should be N(]) directed (26) with subsequent intramolecular rearrangement to the C(3) substitution as temperatures increase. Second, unreacted indole, at reflux temperatures, is directly alkylated at the C(3) position (26). Although slow heating improves yield, the predomi- nant pathway is apparently via the direct 6(3) route since attempts by Fritz (27) to isolate l-indolebutyric acid were unsuccessful. Specific activity of the reaction product was 5545 dpm/pg or 508 pCi/mmole, somewhat below the 833 pCi/mmole expected based on the manufacturers stated specific activity for indole. This activity was adequate for determination of IAA at the level of 50 ng or greater, but for more sensitive methods higher specific activity 14C-IBA would be required. Modifications of the reactions described in order to increase the specific activity of the product should be possible since the y-butyrolactone is already in great excess. Substitution of B-propiolactone for y-butyrolactone would yield 14C-indole-3-propionic acid (26,27). This could serve as an 126 internal standard for use with plant materials which contain com- 14c-IBA. pounds which interfere with Table 1 shows the results of assays of known amounts of IAA after derivatization of the IAA-IBA mixture with BSTFA + 1% TMCS and analyzed with an FID detector and an effluent splitter. The value obtained for IAA is low by 7%, probably due to the non—linearity of the FID detector (23). This problem with FID detectors has been studied (23,24), but for the purposes of this report, a simple cor- rection factor suffices. Multiplying the IAA peak area by 1.07 brings it to agreement with the radioactivity collected, both in amount and relative standard deviation (Table 1). This difficulty with relative detector response for two (even closely related) compounds points to the need to intersperse known samples with the biological material. However, in all cases, during a three month period, while varying sample injection volume by 300% and with sample loading varied from 0.1 to 10 pg the 7% difference in relative detector response was observed. That this difference was, indeed, the result of detector response was confirmed in three ways. First, the amounts of radioactivity yielded the accurate ratios. Secondly, collecting the peaks and analyzing by U.V.282 absorbance confirmed the correct ratios and thirdly, this disparity was not seen in work with the N2T detector. Table 1 also shows the high degree of reproducibility of the method. In practice, multiple injections were always used since the volumes necessary for satis- factory derivatization exceed injection volumes by, at least, 5 fold. 127 Figure 4 shows the recorder tracing resulting from a 2 pl injection of a methylated sample containing 19.7 ng IAA and 29.2 ng IBA per pl and analyzed with the N21 detector. The ratio of IAA to IBA by weight is 0.675 and the molar ratio is 0.783. The peak area for IAA in Figure 4 is 22.8 units and for IBA 29.0 units for an IAA/ IBA ratio of 0.786, which agrees with the calculated molar ratio. Since the N2T detector is believed to respond to the cyanide ion formed in the hydrogen plasma (28) and since one cyanide ion results per indole nucleus, the equal molar response observed is as expected. A plant extract was purified by solvent partitioning, DEAE- Sephadex and BSA-Sepharose and used to compare the relative selec- tivity and sensitivity of the two detectors (Figure 5). Recovery following the purification steps was 30% and the total IAA of corn seedlings after alkaline hydrolysis was found to be 350 ng/g (3, 29). The injection for FID analysis contained 3 pg of IAA as com— pared to the injection for N2T detection which contained 60 ng. The FID sample shows an interfering peak which obscures the IAA peak and a large peak with a retention time somewhat longer than IBA. These interfering peaks are not nitrogen containing and do not interfere with analysis with N2T detection (Figure 5). The NZT detector also proved superior in that it is insensitive to the injection solvents, thus eliminating the solvent tail which made it difficult to integrate peaks on the sloping baseline. Also, the lack of a solvent response allowed the use of increased electrometer sensitivity with these chromatographic conditions. Thus, the NZT detector allows quantitation of samples which are difficult to 128 analyze with an FID detector and offers a 100 f01d increase in sensi- tivity (30 and this paper). By the use of halogenated derivatives of IAA and IBA this method should be adaptable to electron capture (EC) detection. Previously EC has been used for the detection of halogenated deriva- tives of IAA but these methods have not fully corrected for losses and incomplete derivatization (9) or were not useable with plant samples (31). The advantage of EC detection would be a large increase in sensitivity although with loss of selectivity, since any compound which can be acylated after methylation would be detected by EC. We have studied the possibility of using the double isotope dilution method with halogenated derivatives by using repe- titive scan gas chromatography/mass spectrometry. We find that the N—heptafluorobutyryl~indole-3-acetic acid methyl ester can be resolved from impurities in plant extracts after purification by solvent partitioning, DEAE-Sephadex and C18-reverse phase HPLC. The separation requires a 10 ft column and a double derivatization procedure, both of which increase analysis time and reduce sample yield. Impurities in the samples are mainly hydroxy and methoxy cinnamic acids, which are also difficult to resolve from IAA by conventional techniques (29). The presence of large amounts of various lignin acids are serious problems with plant samples and it would appear that the NZT detector, although less sensitive than an EC detector, has advantages in that it requires less sample preparation and is selective to only organic nitrogen or phosporus containing compounds. 129 Several methods for the quantitative analysis of IAA in crude extracts have been proposed. These methods include densi— tometry of thin-layer chromatograms (32), GLC after solvent parti- tioning (33), radio-immunoassay (34), and fluorescence of a chemi- cal derivative (35,36). None of these methods have been compared to chemically rigorous methods of analysis so the reliability of these techniques remain unproven. The method based on the Plieninger reaction (37) is particularly troublesome since it has enjoyed wide usage, apparently without adequate documentation. We have examined this method of analysis using the procedure of Stoessl and Venis (35), the method of Knegt and Bruinsma (36), and a modification we developed using 1 M methanolic KOH as the reaction termination reagent. Our experimental material was 5 day old corn seedling tissue-~tissue which has been extensively studied and which has a high level of endogenous IAA. Measurements were made with an Aminco-Bowman model 4-8202 spectrofluorometer and our standards yielded comparable sensitivity to that reported (36). We utilized the method of quench correction recommended and repeated all assays 4—6 times. One troublesome problem was the reverse addition blank recommended by both Stoessl and Venis (35) and Knegt and Bruinsma (36) which we found increased in fluorescence with time while the samples decreased in fluorescence. This necessitated producing two extrapolation curves for correction to zero time and increased the uncertainty of the final result. By these methods we found at least 1.41 mg/kg of "IAA" in corn seedlings following base hydroly- sis. This is over four times the total IAA concentration in corn 130 seedlings when assayed with isotope dilution techniques, either on a large scale (3,29), by the double standard method, or using d4-IAA as the internal standard (Hall and Schulze, unpublished) with GC- MS-SIM detection. The method we have described using a "double standard" for loss corrections provides an accurate and reproducible method for the quantitation of labile organic compounds when a suitable labeled second standard is available. 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S. and Grigsby, B. H. (1961) Plant Physiol. 66, 354. 133 20. McDougall, J. and Hillman, J. R. (1978) in Isolation of Plant Growth Substances (Hillman, J. R. ed?) p. 1, Cambridge University Press, Cambridge. 21. Caruso, J. L., Smith, R. G., Smith, L. M., Cheng, T. Y., and Daves Jr., G. D. (1978) Plant Physiol. 62, 841. 22. Magnus, V. and Bandurski, R. S. (1978) Plant Physiol. 61(S), 63. 23. Shatkay, A. and Flavlan, S. (1977) Anal. Chem. 46, 2222. 24. Shatkay, A. (1978) Anal. Chem. 66, 1423. 25. Patterson, P. L. and Howe, R. L. (1978) J. Chromotogr. Sci. 16, 26. Remers, W. A. (1972) in The Chemistry of Heterocyclic Compounds, Indoles part one (Houlihan, W. J. ed.), Vol. 25, p. 130, Wiley—Interscience, N. Y. 27. Fritz, H. E. (1963) J. Organic Chem. 26, 1384. 28. Kolb, B., Aver, M. and Pospisil, P. (1977) J. Chromatogr. Sci. 1 53. _’ 29. Bandurski, R. S. and Schulze, A. (1974) Plant Physiol. 64, 257. 30. Swartz, H. J. and Powell, L. E. (1978) Plant Physiol. 61(S), 63. 31. Bittner, S. and Even—Chen, Z. (1975) Phytochemistry 14, 2455. 32. Zimmeggann,H. and Rudiger, W. (1976) Z. Pflanzenphysiol. 16, 33. Wightman, F. (1977) 16_Plant Growth Regulation (Pilet, P. E. ed.), p. 77, Springer-Verlag, Berlin. 34. Pengelly, W. and Meins, F. (1977) Planta 166, 173. 35. Stoessl, A. and Venis, M. A. (1970) Anal. Biochem. 64, 344. 36. Knegt, E. and Bruinsma, J. (1973) Phytochemistry 12, 753. 37. Pliergiingggi H., Muller, W. and Weinerth, K. (1964) Chem. Ber. _’ Fig. 2 Fig. 3 Fig. 4 Fig. 5 134 Legends for Figures Reaction scheme for the synthesis of 14C-[2-ringJ-indole- 3-butyric acid from 14C-[2-ring]-indole and y-butyro- lactone in strong base. 14 Ultraviolet absorption spectrum for the synthetic C- [2-ring]-indole-3-butyric acid after Sephadex LH-20 chromatography. ' 70 ev electron impact mass spectrum of the synthetic A 14C-[2-ring]-indole-3-butyric acid methyl ester. Evident ‘ is the molecular ion at 217, the m-31 from loss of [-O-CH3] at 186, the m-74 with loss of [-CHz-COOCH + H] at 143, 3 and base peak at 130 from the B-cleavage fragment. Nitrogen specific thermionic detector recorder tracing of a mixture of IAA and IBA as the methyl esters. The injec- tion volume was 2 pl and contained 19.7 ng IAA and 29.2 ng IBA per p1. Methylated sample from corn seedlings analyzed with the flame ionization detector (FID) and the nitrogen specific thermionic detector (N2). The injection for the FID con- tained 50 times the sample load as was injected for the thermionic detector analysis. 135 .2259. wt .55 E $.12 cm B< Am .9: MN ._mo ngmvcmpm mmwmm mm.o.H a.m_ Fmo.o m_m.o mm=Fm> cam: “comm m.m— Nvo.o m.mm m.w¢ mmm.o mmmm mum o emvmm F.n_ owm.o a.mm ¢.om wpm.o omom wmm m mpwmm o.m_ Pmo.o N._m a.mm me.o wmqm omm q ommom ¢.w_ mmo.o m.mo N.m¢ vom.o mF—N mmv m ommmm ¢.w_ Nmm.o m.¢m m.mm mmm.o womm mmm N memm m.wF mqo.o a.mn m.x¢ wF~.o “NFN mow _ < umum_:o_mu owpmm mmc< owpmm .cowpompmn cowpmwwcow wEMFw vcw mush &F + wcmv mcwm: mcowpuwwcw w>apwpmamg spas cmcwmpno mas—m> Faucmswgmaxu11.~ mam<~ CONCLUSIONS Hall and Bandurski (1978) found that when radioactive IAA was applied to the endosperm of corn seedlings radioactivity would move to the shoot, but only 2% of the radioactivity was in IAA or IAA esters. Most of the IAA which survived movement to the shoot was esterified. These results were extended by Nowacki (see Bandurski, 1978) who showed that ‘4 C-indole—3-acetyl-myo-inositol migrated from the seed to the shoot in much larger amounts than labeled IAA and that very little destruction occurred even though 14c-IAA was identical to the the ratio of free and esterified endogenous compliment. These results suggested a protective role for the IAA conjugates which could be important for the hormone while in route. Likewise, other hormones are found as conjugates in the transport stream or have been shown to be transported as conjugates (Sembdner et al., 1968; Van Staden and Dimalla, 1978) and they may also therefore be protected from degradation (see discussion in Wang et al., 1977). In addition to protection, the bound compounds could also play an important role in defining the destination of the transported hormone--a kind of biological "zip code" where, as in the postal service, lack of the "zip code" results in loss of the hormone. 141 142 Although the contribution of peroxidative degradation to overall auxin destruction is probably only a fraction of the total, it does serve as a convenient model and could be especially import- ant in transport since at least some peroxidases are localized in the cell wall region (see review by Lamport, 1970). Thus, the demonstration that conjugation protects the hormone from oxidative degradation (Cohen and Bandurski, 1978) may be important in under- standing the function of these compounds. Early notions that conjugation was a detoxification mechan- ism (Gordon, 1954; Andreae and Good, 1955; Aberg, 1957) may be incorrect since no degradation route is known that does not first release the free hormone. For a compound to be a detoxification product it must be secreted, sequestered, or destroyed and these compounds do not seem to function in this manner. A more likely role is for these compounds to function as a regulatory mechanism for the control of free hormone levels. This contention is sup- ported by the observation that conjugate levels do not remain static during germination, as would be expected for sequestered detoxification products, but decline at a linear rate (Ueda and Bandurski, 1969; Cohen, unpublished). Similar utilization of bound forms has been shown for other hormones (cf. Hiron and Wright, 1973; Sembdner, 1974; Davey and Van Staden, 1977). Also, as the bound auxins decline in amount, the levels of free hormone remain cone stant, at an apparently regulated level (Ueda and Bandurski, 1969; Cohen, unpublished). In a similar manner, but in reverse, mature 143 soybean callus cultures control the level of free 2,4-D by conju- gation (Davidonis et al., 1978). The regulatory role of the IAA esters was demonstrated most convincingly by our results (Bandurski et al., 1977) which showed that the ratio of bound to free IAA can be perturbed by environmental inputs such as a light stimulus. This is the first instance in biology for which we have evidence for hormonal levels being con- trolled by the formation and hydrolysis of a covalent bond. In addition it is the first experiment on light effects on growth and tropisms in which the hormone has been identified by accurate physi- cal methods. It is, therefore, an important step in understanding in precise chemical terms the nature of the light-growth process and may lead to new insights which will explain the discrepancies between the experimental data from several groups and the Cholodny- Went theory of tropisms (see literature review). The experiments on the effects of light on hormone levels required 600-2000 grams of tissue for each determination using the techniques then available. In order to study more complex biologi- cal phenomena better methods had to be developed. The chemical synthesis of indole-3-acety1-myo-inositol was an important step in this process since it will allow us to study the conjugation process as well as transport and environmental effects. The unlabeled pro- duct is important for reverse isotope dilution analysis where radio- labeled conjugates might be formed by tissue or enzyme extracts (cf. Michalczuk and Bandurski, 1979) in small amounts. The availability 14 of high specific activity C-indole-3-acetyl-myo-inositol has 144 already been used for transport studies as indicated above and for studies of metabolic turnover (Epstein and Bandurski, 1978). A prior synthesis of the 2-0-indole-3-acetyl-mya-inositol isomer was accomplished by Tate (cited in Nicholls, 1967) by condensation of pentabenzyl-myo-inositol-2-ol with IAA followed by hydrogenation; however the yields were very poor. Chemical synthesis of other hormone conjugates has been previously reported. The 1-0 isomer of the glucose ester of IAA was synthesized by Keglevic and Pokorny (1969) by reacting 2,3,4,6- tetra-O-benzylglucopyranosyl chloride with the silver salt of IAA followed by hydrogenation. Glucose esters of other hormones have been accomplished by similar procedures or by using the peracety- lated sugar (Schreiber et al., 1969; Hiraga et al., 1974a; Schneider, 1974; Lehmann et al., 1975; Cowley et al., 1978). Synthesis of the amino acid conjugates has been accomplished by several methods (see Weiland and Horlein, 1955; Good, 1956; Weller and Sell, 1958; Armstrong et al., 1958; Hart et al., 1970; Mollan et al., 1972; Hattori and Marumo, 1972; Lischewski et al., 1974). However, our 14C-indole-3-acetyl-myo-inositol (Nowacki report on the synthesis of et al., 1978) is the first chemical synthesis of a radiolabeled bound plant hormone and is one of the rare examples of a successful high specific activity radiochemical synthesis at the micromole scale. Previously, radiochemical methods have been used to follow enzymatic synthesis in vitro and this has resulted in small amounts of radiolabeled products which were used to monitor the reaction (Hutzinger and Kosuge, 1968b; Kopcewicz et al., 1974; Muller et al., 145 1974; Entsch and Letham, 1979) but these methods produced yields too low to allow the product to be isolated and used in subsequent investigations. Although it appears that an equatorial isomer of the inosi- tol esters is produced in the enzymatic synthesis (see Bandurski, 1978; Michalczuk, personal communication), a mixture of isomers is found in corn seeds even when relative mild extraction conditions are used and acyl migration occurs rapidly even at neutral pH (Nicholls, 1967). This, as well as the knowledge that inositol esters substituted at multiple positions are found (Ehmann and Bandurski, 1974), suggests that the plant has found some way to deal with multiple isomeric forms. How this is accomplished must await characterization of those enzymes responsible for release of the free hormone. The rapid method for the analysis of the inositol esters has resulted in improved procedures for evaluation of products produced by enzymatic reaction and has resulted in the identifica- tion of indole—3-acetyl—myo-inositol from rice when older, slower methods had failed (Michalczuk and Bandurski, 1979; Hall and Bandurski, 1979). 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