THE BIOCHEMISTRY AND PHYSIOLOGICAL IMPLICATIONS OF lNDOLE-S-ACETEC ACID IN THE DEVELOPING PEACH (PRUNUS PERSICA) SEED Thesis for The Degree of Ph. D. MICHIGAN STATE UNIVERSITY Rager WiIIEam Réfzerf 19:56 j ‘ ran—Im-‘q ‘masm I I: fl yl A Y IV'II(TI13‘-f:1:1 5:1“11’6 University I... w This is to certify that the thesis entitled THE BIOCHEMISTRY AND PHYSIOLOGICAL IMPLICATIONS OF INDOLE-B-ACETIC ACID IN THE DEVELOPING PEACH (PRUNUS PERSICA) SEED presented by Roger William Ritzert has been accepted towards fulfillment of the requirements for ‘? PhoD. degree in BiOChemiStry AMM-M Major professor Mm 35; we 0-169 ABSTRACT THE BIOCHEMISTRY AND PHYSIOLOGICAL IMPLICATIONS OF INDOLE-B-ACETIC ACID IN THE DEVELOPING PEACH (PRUNUS PERSICA) SEED by Roger William Ritzert Various phases of fruit growth and development have been suggested to be under the control of endogenous hormones. A previously unidentified auxin has been shown to be present in highest concentration during cytokinesis of the endosperm cells in immature peach (Prunus persica) seeds and thus has been implicated as being functional during this stage of development. The enhanced effect by naphthaleneacetic acid as a thinning agent during cyto- kinesis furnished further evidence that there was an increase in the endogenous auxin supply. Previous work has suggested, but not confirmed, that indole- 3-acetic acid is the auxin which may be implicated in cytokinesis. A mechanism for the control of its relative content was suggested from 12 3112 studies which showed a decrease in the ability of the immature peach seeds to decarboxylate indole—B-acetic acid during cytokinesis. Past knowledge of the indole—B-acetic acid oxidase system has shown that orthopolyphenolic compounds, which occur widely in plants, are inhibitors of the oxidative process. Regula- tion of possible indole-3—acetic acid destruction by inhibition of the enzyme was suggestive, but neither the enzyme nor an inhibitor had been previously demonstrated in homogenates of the immature seed tissue. Roger William Ritzert Indole-B-acetic acid was identified as the acidic auxin in the immature seeds of Redhaven peach fruit whose increase in concen- tration correlated with the time of cytokinesis of the seed endosperm. Identification of the auxin was made on the basis of its biological activity, mobility on paper and thin-layer chromatograms, and ultraviolet absorption spectra. An additional acidic auxin, not previously described as being present in the peach, was detected in the immature seed extracts. The compound had a Rf 0.80-1.00 in butanol-ammonia water (100:3:18) and displayed biological responses typical of auxins and colorimetric reactions typical of indoles. Evidence was also obtained for the presence of a neutral auxin. An indole-B—acetic acid oxidase system was detected and characterized in the soluble fraction of peach seed extracts. Enzyme activity could be detected only after treatment of the extract with Tween 80 to disrupt possible protein—tannin complexes, and separation of the protein fraction by passage through Sephadex 6-25. The enzyme had a pH optimum of 4.5 to 5.0 and required both Mn++ and 2,4-dichlorophenol for optimum activity. The product of the reaction was identified from the absorption spectrfim of the reaction mixture as 3-methy1eneoxindole. A polyphenol, catechin, was identified by chromatography in seed extracts. An authentic sample of catechin was shown to be a non-competitive inhibitor of the peach oxidase enzyme system. The inhibitory action of catechin on indole-3-acetic acid oxidase is considered as a possible mechanism by which the concen- tration levels of indole-B-acetic acid may be adjusted during fruit Roger William Ritzert maturation. The possible complex nature of the unidentifed acidic auxin is also discussed. THE BIOCHEMISTRY AND PHYSIOLOGICAL IMPLICATIONS OF INDOLE—B—ACETIC ACID IN THE DEVELOPING PEACH (PRUNUS PERSICA) SEED By Roger William Ritzert A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1966 ACKNOWLEDGEMENTS The encouragement and guidance of Dr. H. M. Sell throughout the author's graduate program is gratefully acknowledged. Words of appreciation are extended Dr. M. J. Bukovac for many stimulating discussions and use of laboratory facilities for portions of the study. The support and interest of the author's parents throughout his educational experience will always be remembered. The love, encouragement, and often sacrifice of the author's wife and children during the doctoral program has helped to make this a most rewarding experience. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . . . . . . . . . . Role of Auxins in Fruit Development. . . . Other Growth Regulators in Fruit Development . Indole-B-Acetic Acid Oxidase . . . . . . . Biochemical Aspects of Auxin Action. . . . . METHODS AND MATERIALS. . . . . . . . . . . . . . . . Identification of Auxins . . . . . . . . . Extraction and purification . . . . . . Chromatography. . . . . . . . . . . Preparative thin-layer chromatography . Coleoptile straight-growth assay. . . . Identification of Catechin . . . . . . . . Indole-B-Acetic Acid Oxidase . . . . . . . . Preparation of soluble enzyme fraction. Enzyme assay. . . . . . . . RESULTS. . . . . . . . . . . . . . . . . . . Auxin Identification . . . . . . . . . . . Chemical. . . . . . . . . . . . . . . . Bioassay. . . . . . . . . . . . . . . Catechin Identification. . . . . . . . . . Indole-B-Acetic Acid Oxidase . . . . . . L DISCUSSION . . . . . . . . . . . . . . . . . . . . . SUMMARY. . . . . . . . . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . iii 11 13 15 16 16 20 20 22 24 24 25 26 27 27 30 38 46 57 61 TABLE II. III. IV. LIST OF TABLES Chromogenic Reagents for Detection of Indoles . Characteristics of Auxin Detected in Immature Peach Seeds. . . . . . . . . . . . . . . . Properties of DMAC—Positive Components in Seed Extract. I O O O O O I O O O O O O O O O 0 Evidence for Catechin in Immature Peach Seeds . iv— PAGE 21 28 29 41 4. 5. 6. 10. 11. LIST OF FIGURES PAGE Scheme used for extraction of auxins from peach seeds. Details are given in text. . . . . . . . . . . 18 Biogram resulting from separation of extract of 50 mg of seeds collected on June 11, 1964, (cytokinesis) with isoprOpanol-water . . . . . . . . . 31 Biograms resulting from separation of extracts of 50 mg of seeds collected (A) before, (B) during, and (C) after cytokinesis with butanol-ammonia-water . 33 Biograms resulting from separation or extracts of 50 mg of seeds collected (A) before, (B) during, and (C) after cytokinesis after separation of non-acidic components. Method used is given in text. . . . . . . 36 Comparison of IAA content to fruit growth and especially cytokinesis. Fruit growth was measured by pericarp diameter and IAA content by bioassay of duplicate biogram sections of 0.3 to 0.4 Rf . . . . 39 Effect of catechin on IAA induced growth of Avena coleoptiles. Molar concentrations of catechin are designated for the respective curves . . . . . . . . . 42 Effect of catechin on buckwheat root growth. . . . . . 44 Elution of soluble protein extract and IAA oxidase activity from Sephadex G-25 column. Oxidase activity measured colorimetrically as moles x107 destroyed /ml/30minutes.................... 47 Optimum pH conditions for IAA oxidase activity. IAA destroyed /ml was measured after 2, 4, and 6 hours. I O O O O O O O O O O O l O O I O O O O I O O O 49 Ultraviolet absorption spectra of reaction mixtures showing cofactor requirements for the peach IAA oxidase. Curve 1, complete reaction mixture after 1 minute; curve 2, same after 40 minutes; curve 3, same after 10 hours; curve 4, same minus 1 x 10-4 M MnC12 after 10 hours; and curve 5, same minus 1 x 10-4 M 2,4-dichlorophenol after 10 hours. . . . . . . . . . . 51 Rate of 3-methyleneoxindole formation catalyzed by peach IAA oxidase and its inhibition by catechin. The Lineweaver-Burke plot (inset) was prepared utilizing (1) no catechin, (2) 2 x 10” M catechin, and (3) 4 x 10-7 M catechin. . . . . . . . . . . . . . 53 INTRODUCTION INTRODUCTION In addition to the many hormonal functions attributed to auxins in controlling plant growth, hormones are known to be functional in various phases of fruit growth and development. Auxins have been implicated in cytokinesis in immature peach seeds and generally occur in highest content during cytokinesis. Further, naphthaleneacetic acid is most effective in inducing fruit abscis- sion during this critical time. No definite determination of the auxin which may be involved in this process has been reported (48, 52). Although indole-B-acetic acid is generally implicated as the principal plant auxin, there are numerous reports that derivatives of indoleacetic acid are present in some plant tissues. Recent evidence has also indicated the occurrence of a non-indolic auxin in several plant species, including peach fruit. Thus, it was of interest to establish if there was a relationship between endogenous auxin and cytokinesis and to identify the auxin involved. Assuming that indole-B-acetic acid is the auxin in question, a mechanism for the control of its relative content was suggested when in 3123 studies showed a decrease in the ability of the immature peach seeds to decarboxylate indole—B—acetic acid during cytokinesis. Thus, a control of the rate of breakdown, not synthesis of the auxin, was suggested. The control mechanism could be mani- fested through regulation of indole-B—acetic acid oxidase enzyme synthesis or through control of enzyme activity by an inhibitor. I\) 3 Inhibition of the enzyme is accomplished by orthopolyphenolic compounds, which are widely distributed in higher plants. Two objectives were established, namely: To define the nature of the auxin or auxins present in the developing seed, and to describe biochemical mechanisms which may be responsible for regulating the levels of auxin present. LITERATURE REVIEW LITERATURE REVIEW Role 2: Auxins in Fruit Development Auxins are defined as "compounds characterized by their capacity to induce elongation in shoot cells" (93). There is much evidence for the participation of natural auxins in fruit develop- ment. The historical development of these concepts will be briefly outlined. Nitsch describes a fruit as consisting "of the tissues which support the ovules and whose development is dependent upon the events occurring in these ovules" (64). The growth of the fruit can be summarized as two main growing periods: that portion before anthesis during which growth proceeds mostly by cell division, and a post- fertilization phase which is characterized essentially by cell enlargement. Post—fertilization, to which further discussion will be ‘directed, is composed of three stages of growth as described by Connors (6) for the peach (Prunus persica). Stage I is characterized by rapid growth of all parts of the ovary and its contents, except for the endosperm and embryo. Stage II displays very little growth of the ovary, lignification of the endocarp, and rapid growth of the endosperm and embryo. Fruit maturity is achieved following rapid growth of the mesocarp during Stage III. Since the increase in volume during the growth of fruits is mainly by cell enlargement and auxin is known to act by controlling ‘cell enlargement in other tissues, it has often been assumed that such was the controlling factor in fruit growth (8). There appears 6 to be a correlation between seed development and distribution, and the ultimate size and shape of the fruit. 'Tukey (92), studying several species of Prunus including the peach, noted a relation between point of attachment of the ovule and development and size of the fruit. This would suggest the involvement of a stimulus passing from the developing ovule through the vascular system into the adjacent tissue. Additional indications that the seed is the source of auxin was presented by Nitsch (63) in studies with the strawberry. The weight of the fleshy receptacle was approximately proportional to the number of achenes (fertilized ovaries) contained thereon. Removal of the achenes from some areas resulted in failure of the receptable tissue to grow. The achenes were found to contain free auxin, while the receptacle tissue contained none. Gustafson (28) ‘had dimilarly shown that the auxin content of ovules and deveIOping seeds of the tomato fruit is much greater than that of other fruit parts. This led to his early suggestion that the seeds produce auxin which influence the growth of the surrounding tissue. Further evidence for the involvement of auxins in fruit ‘ growth can be found in several instances where relationships between auxin content and growth of the fruit are reported.' Relations of this type are based on the auxin content of extracts as measured by one of the common auxin assays, straight-growth or curvature. Luckwill (54, 55) demonstrated that the appearance of a hormone in the apple seed coincided with development of the endosperm. He further showed that natural fruit abscission could be correlated to low hormone content and that this occurred after developmental 7 stages which would require hormone. Wright (94) was able to show that high levels of an auxin in the currant coincided with cell division. Fluctuations in total auxin content were observed by Nitsch, 23 al. (70) in seeded grapes and were correlated with meristematic activity of the seed. In the seedless grape, there was a correlation with cell division. In studies on parthenocarpic and non—parthenocarpic figs, Crane at 31. (9) did not observe a correla- tion between free auxin in the fruit and growth rate. A requirement for free auxin in leaves for cell division has been substantiated(3®. Stahly and Thompson (87), using ovules of the developing Halehaven peach, reported the existence of three auxins, each with its own pattern of fluctuation. During the periods when the two 'major auxins were at their highest levels, the fruit were in a period of suspended growth. These authors suggested that the high auxin content was acting to inhibit cell enlargement, and thus growth at this stage. Crane (8) has pointed out that this could not be true since parthenocarpic fruit exhibited the same growth pattern. Powell and Pratt (77) showed two distinct periods of high hormonal concentration during the maturation of three peach varieties. The first wave of auxin was correlated with the time of cytokinesis in the seed. Lombard and Mitchell (52) noted increases in levels of both an acidic and a neutral auxin 43 days after bloom in seeds of the Redhaven peach. According to extensive data compiled by Ieuty (48), this period is most likely the time of cytokinesis. He also showed that the pericarp diameter measures 30 mm at this stage and that maximum fruit abscission was induced by exogenous auxin (NAA) 8 applied at this time. These above results point to the importance of some'auxin'or‘auxins during cytokinesis. There is considerable evidence that indolee3-acetic acid (IAA) is the principal auxin in higher plants. Two excellent reviews by Bentley (1, 2) list numerous species and the nature of the auxins which have been detected in them. The auxins present in some fruit tissueS'will be discussed further because of their pertinence to the subject.' A notable flaw in relating these auxins to fruit develop- ment is the failure by most workers to utilize bioassays which are as close as possible to the type of growth being studied (45). In most instances, straight-growth assays of paper chromatogram sections accompanied by chromatographic characteristics have been used for identification of the auxins, usually indolic in nature. Luckwill (56) identified three acidic auxins in the leaves and fruits of the apple, but none of the auxins appeared to be the same as the common indole compounds, particularly IAA. The absence of IAA and the presence of a non-indole auxin was shown by Luckwill and Powell (57). Other reports (14, 15, 36, so, 90) indicate that 'IAA and several of its derivatives are present in some varieties of apple. The ethyl ester of IAA (EIA) was extracted by Teubner (90) from apple endosperm using ethanol, so the compound might have been 'IAA; There is also evidence that IAA is present in the currant (94) and grape (70). Stahly and Thompson (87) showed the apparent presence of IAA and two other auxins which are probably indolic. It should be noted that their detection by chromogenic reagents was 9 transient, but this may very well be due to low levels of the compound. Khalifah, gt a}. (41) have recently reported the presence of a non-indolic auxin in young citrus fruit whose presence can be correlated with the development of the orange (40). These workers have also noted the presence of "citrus auxin" in the young fruit of the date, fig, plum, and peach (49). The physiological role of this auxin, especially in tissues where IAA has been detected is not immediately clear. Other Growth Regulators in Fruit Development In addition to auxins, there is evidence that both kinins and gibberellins are involved in regulation of plant growth. The general aspects of these two substances will be reviewed only as they relate to fruit growth and development. Thorough reviews on gibberellins (74) and kinins (58) have appeared recently. Gibberellins are known for their ability to stimulate cell division and cell elongation. There is some evidence to implicate a requirement for gibberellins in fruit growth. One line of evidence is the ability of gibberellic acid (6A3), in particular to set parthenocarpic fruits (8), including the peach (lO). Gibberellins A3 and A4 have been shown by Bukovac (4) to set parthenocarpic fruit of the apple. Phinney et al. (75) have indicated that young seeds, including almond, apricot, and plum seeds, are rich sources of gibberellinelike activity. Apple endosperm has been shown by Nitsch (65) to contain gibberellin-like substances. Three gibberellin-like 10 substances, two of which appear to be A1 and A9, have recently been detected in citrus fruits (40). Although increased levels of gibberellin-like substances seem to accompany growth of the seed, no relationship has yet been established between fruit growth and levels of these substances as there exists for auxins (8). These findings, when considered with the knowledge we have of auxins in fruit growth, led Bukovac (4) to suggest that no single stimulus can be considered by itself in relation to fruit growth and that several factors alone or interacting might be responsible. Kuraishi and Muir (42, 43) have shown that treatment with gibberellic acid causes an increase in diffusible auxin from stem apices.‘ Other work demonstrates that gibberellin treated tomato ‘ovaries produce the same quantity of diffusible auxin as pollinated ovaries (86). The fact that both auxins and gibberellins are probably in the seeds of most fruits may suggest an interrelationship of the'two, with gibberellin perhaps being the controlling factor of auxin synthesis. Evidence for this control system has been suggested by Sastry and Muir (85), who found that gibberellic acid stimulated the conversion of tryptOphan and other IAA precursors to IAA in apical 53323 segments. The work with kinetin, a synthetic kinin, exhibited that kinin~induced cell division takes place only in the presence of IAA (58). Very little is known about the natural kinins in fruit tissue except from the scattered reports of kinin~like activity in extracts of these tissue. The first observation of a kinin in young apple fruits (later demonstrated to be similar to that found in coconut ll milk [98]), was made by Goldacre and Bottomley (22). Likewise, plum fruitlets (46, 47) yielded a kinin whose properties appear to be similar to an unknown purine crystallized from an extract of sweet corn (46). Lavee (44) noted kinin-like activity in extracts of peach fruitlets. Kinins were found by Powell and Pratt (78) in extracts of the embryo and endosperm of the immature peach seed. The kinin activity which requires IAA, may be more abundant in the endosperm than in the embryo suggesting a possible interaction in cytokinesis. Nitsch and Nitsch (68) have also shown kininwliks activity in tomato fruit. Most interesting results have been obtained recently by Crane and van Overbeek (11) in inducing the parthenocarpic growth of fig fruit with a synthetic kinin. A possible interrelationship of auxins, gibberellins, and kinins gains momentum from this experimext since identical parthenocarpic fruit can be set with either auxin or gibberellin. As stated earlier, in all cases reported, IAA or natural auxin are necessary for kinin-induced cell division. Indole—B-Acetic Acid Oxidase The number of reports concerning the systems for destroying IAA in plant tissue are voluminous. Reviews by Ray (81) and Hire (29) will serve as a broad background on this subject for those unfamiliar It will suffice to say that IAA can be rapidly destroyed by an oxidasevperoxidase system extracted from various tissue. The course of disappearance of IAA may be followed manometrically (O? or CO2), colormetrically (84), or spectrophotometrically (82). [he enzyme has 12 been suggested by Ray (83) to be the same as the peroxidase of Omzhelie flavida. Horseradish peroxidase will also carry out the destruction of IAA (32, 33). It has recently been shown that the horseradish peroxidase system oxidizes IAA (I) to oxindole-B- carbinel (II) and then to 3~methyleneoxindole (III). The same product: were detected from a riboflavin-catalyzed photooxidation of IAA (18). 3-Methyleneoxindole is known to be a potent growth inhibitor of higher plants and microorganisms (18, 88). Inhibition of grOWth may be due to the binding of 3-methy1eneoxindole to the sulfhydryl groups of enzymes (88). H ‘ HZCOOH CH2OH =CH2 a "é N x. N *0 \ N SO H H H I II III Monophenols are known to be catalytic to the destruction process (29, 81), with 2,4odichlorophenol commonly being used to assay the enzyme (23). The metallic requirement is satisfied by manganous ion (31). Several naturally occurring monophenols have been shown to serve as cofactors in both in_vitro and in vivo (96) systems. Meet polyphenols with adjacent phenolic groups are inhibitors of the IAA oxidase system (29, 81). Naturally occurring inhibitors have been isolated from tissues known to possess IAA oxidase systems. F€¥U11¢ acid was shown by Gortner and Kent (26) to be a naturally occurring inhibitor of the pineapple oxidase system. An inhibitor of 13 the oxidase in light-grown cotton has been demonstrated, but not characterized (59, 60). Mumford 3: al. (61) have isolated a kaemferol derivative from the pea plant which acts as an inhibitor of the oxidase. This inhibitor, however, does not have the expected adjacent phenolic groups. A synergistic effect by chlorogenic acid, an inhibitor of the oxidase, was noted on IAA induced curvature in £2323 coleoptiles (91). The same was not true for indole—B-butyric acid and naphthaleneacetic acid. Momophenols (cofactors for IAA oxidase) had the reverse effect. Galston (19) has related inhibitor levels to the photo- morphogenesis of the pea plant. Thus, a direct relationship between inhibitor levels and quality and quantity of light is suggested. In addition, there appears to be an effect of light quality on free IAA content in bean plants (17). Inhibitor levels may be controlled by light, thus affecting IAA content and ultimately growth. Donoho gt 3}. (13) have detected IAA oxidase activity in Redhaven peach seeds using in_vivg techniques. The decarboxylation of carboxyl labeled IAA was used as a measure of activity. The oxidase activity at the time of cytokinesis was lower than other periods suggesting the possibility of a control mechanism for IAA. Kefford 3: al. (35) have suggested that IAA oxidation may play a role in IAA growth regulating functions. Biochemical Aspects of Auxin Action Our present level of knowledge in the area of molecular biology has Opened the road for severalllaboratories to apply the methodology to the biochemical aspects of auxin—induced growth. 14 Evidence that auxin-induced cell enlargement is accompanied by protein synthesis was reported by Nooden and Thimann (71, 72) and confirmed by Key (37). The uptake of glycine, presumably into protein, is enhanced by IAA in an EB.X1£EE system (16). The synthesis of DNA-like RNA, probably messenger RNA, is necessary for the growth of excised plant tissue (20, 37, 38). Further, the synthesis of RNA is enhanced by auxin (39). These results suggest that perhaps the auxin induced response controls synthesis of the message for protein synthesis. Whether this is a specific protein or not is not known. Some possible significance may be found in the observation that IAA can be bound to the RNA fraction, but only in the presence of peroxidase (20, 35). IAA can be conjugated to a protein (95). METHODS AND MATE RIALS METHODS AND MATERIALS Identification of Auxins Peach fruit (Prunus persica, cv. Redhaven) were collected at periodic intervals between fertilization and pit hardening. The 'seeds were removed from the fruit immediately after harvest and cropped into containers packed in dry ice. Additional fruits were placed in plastic bags and stored at -20°. The frozen seeds were lyophillized on a Virtis freeze-drier and then ground using a Virtis homogenizer. The ground seeds were stored at -10° in closed containers. Extraction and purification Preliminary experiments were designed for the purpose of detecting IAA in the seed extracts. Five grams of dried, ground seeds were covered with 125 ml of dry peroxide-free ethyl ester and kept at 4° for 1 hour. The mixture was filtered and the above extraction repeated twice. The combined filtrates were evaporated to dryness for further purification by one of the following methods. The residue was taken up in 50 m1 of ethanol and passed through a column of acid activated alumina (51) (0.9 cm x 15 cm) to adsorb the acidic components. The column was washed with 50 ml of ethanol and eluted with 25 m1 of 0.1 N KOH. The basic solution was acidified with l N H2804 to pH 2.8 and the acids extracted from the aqueous solution with ether. The concentrated ether extract was used for identification by chromatographic means. 16 17 The other method used is essentially that described by Nitsch (66). The residue was taken up in 25 ml of dry acetonitrile and extracted with dry hexane until the hexane remained colorless. The acetonitrile fraction was taken to dryness and the residue dissolved in 25 m1 of ether. The other solution was extracted three times with 10 ml of 5 per cent NaHCO3. The remaining ether fraction constitutes the non-acidic components. The NaHC03 solution was acidified to pH 2.8 with 6 N HCl. Extraction of the acidified solution with three portions of ether gave the acid fraction which was used for chromatography. Both of the above clean-up procedures are designed to remove lipids which interfere with bioassays and consistent mobility of the auxins on paper. These were useful for detecting and identifying IAA in the extracts, but did not offer consistent quantitative results as measured by the straight-growth assay. A method which offered the following advantages was thus developed: (1) remove lipid material, (2) preserve extracted auxins unaltered, (3) give qualitative and quantitative reproducibility, and (4) speed. For these reasons, the procedures as outlined in figure 1 were utilized for the major portion of the experiments. One or two grams of dried seeds were extracted with 25 m1 of ethyl acetate at 4° with constant stirring for 20 hours. The mixture was filtered through a sintered glass filter and the residue extract— ec two more times for 1 hour. The combined extracts were reduced in volume to 3—4 ml and mixed with 0.5 g of powdered cellulose. The solvent was completely removed under a stream of N2. The cellulose 18 Figure 1 Scheme used for extraction of auxins from peach seeds. Details are given in text. 19 LYOPHILLIZED SEED ETHYL ACETATE EXTRACTION EXTRACT CELLULOSE COLUMN ELUTE WITH 80% MeOH AQUEOUS MeOH SOLUTION TO DRYNESS MeOH MeOH SOLUTION / \ PURIFICATION AND BIOASSAY IDENTIFICATION 20 with sample was placed on a column of powdered cellulose (0.9 cm x 10 cm) and the column eluted with 50 ml of 80 per cent methanol. The total eluent was taken‘to dryness and the residue taken up in 1 ml of methanol. Portions of this solution were used for bioassay, and for purification and identification. Chromatography Paper chromatography was carried out on Whatman 1 mm paper (9-1/8 in. x 24 in.) using the Drummond paper chromatography apparatus. The paper was developed using the ascending technique after equilibration with the solvent. Auxins were detected using the 51323 coleOptile straight-growth assay on sections of chromatograms on which extracts of 50 mg Of seeds were separated. Separations using thin-layer chromatography were effected using 0.25 mm layers of silica gel G or H. The chambers were kept saturated prior to and during development of the layers using paper dipped in the solvent and adhering to the side of the chamber. The Chromogenic reagents used on various occasions are listed in Table I. The DMAC reagent was used most frequently because of its stability and sensitivity. The Prochazka reagent was useful for detecting low levels of indoles on thin layers. Preparative thin-layer chromatography Portions Of the stock solution Of the extract representing about 0.5 g of dry seeds were spotted across a thin layer plate. The layer was developed with either chloroform-acetic acid (95:5 v/v) or iSOprOpyl ether-acetic acid (95:5 v/N) using IAA as a marker. After 21 Table I. Chromogenic Reagents for Detection of Indoles Reagent Reference Preparation Comments p-Dimethylamino- 30 l g pedimethylaminoa Sensitive, turns cinnamaldehyde cinnamaldehyde, red 12 hours (DMAC) 50 ml ethanol, after applica- 50 m1 6 N HCl tion p-Dimethylamino- 3 0.5 g p-dimethyl— Good, less benzaldehyde amino-benzaldehyde, sensitive (Ehrlich) 1 ml 12 N HCl, 100 ml ethanol FeCljéHC104 25 1 ml 0.5 M FeC13, Most sensitive for (Salkowski) 50 ml 35% HC104, IAA over other 50 ml ethanol indoles Prochazka 5 10 ml 35% formalde- Heat 5 minutes at 100°; faint colors become fluorescent under long wave U.V. lamp, very sensitive hyde, 10 ml 12 N HCl, 20 ml ethanol 22 development, the layer was covered except for that portion with the marker, and sprayed with DMAC reagent or Prochazka reagent. That portion of the layer correlating to IAA was removed and eluted with methanol. The eluent was reduced to 0.1 ml and 0.01 ml chromato- graphed on thin layers of silica gel G'or H for characterization. Additional areas producing indole-positive colors were removed and handled similarly. Coleoptile streightggrowth assay The assay used is essentially that described by Nitsch and Nitsch (69). The green light source was supplied by green fluores- cent tubes Wrapped with two layers each of green and amber acetate sheets. ‘Azggg'seeds, cv. Torch, were soaked in tap water for 2 hours in the dark. The seeds were placed embryo up on moist filter paper spread over a porcelain plate. The plate was laid in a culture dish in sufficient glass-distilled water to keep the paper moist and covered. After germination at 25° for 24 hours, the seeds were exposed to 2 hours of red light (photographic safe-light) to inhibit elongation of the first internode and returned to the dark. The coleoptiles were harvested when they were 20-25 mm in length, usually 72 hours after planting; In later experiments, the seeds were spread at random after soaking on a layer of moist vermiculite, germinated under red light at 25° for 18 hours, covered with l/4-inch of vermiculite, and placed in the dark at 25°. The coleOptiles were harvested as described before. A 4.5 mm section of the coleoptile was removed 3—4 mm below the apex. Sections were floated on Mn804 (1 mg/l) disSolved in 23 glass-distilled water for 3 hours. Test solutions were made'up in a pH 5.0 phosphate-citrate bUffer (1.794 g KQHPO4 and 1.019 g citric acid monohydrate) containing 2 per cent sucrose and 0.1 per cent Tween 80. Bioassays of paper strips were made by adding 1 ml of buffer to a tube with a portion of the paper representing a 0.1 Rf unit. Sections were taken from -O.2 Rf to 1.0 Rf. The -O.2 to 0 Rf section served as the control. Ten coleoptile sections were added to 1 ml of solution in an 18 mm x 150 mm tube. The tubes were rotated on a horizontal axis at 1 rpm in the dark at 25° for 20 hours. The sections were then measured to the nearest 0.2 mm using a photo- graphic enlarger. Identification of Catechin Ten grams of ground, dried seeds collected on June 12, June 20, and July 1, 1962, were extracted with 200 ml of 50 per cent acetone at 4° for 24 hours. The mixture was filtered and the residue reextracted for 24 hours. A drop of the combined extracts spotted on paper produced a green color when sprayed with the DMAC reagent. The acetone was removed in vague and the remaining aqueous solution saturated with NaCI. The NaCl saturated solution was extracted twice with 100 ml of acetone and the combined acetone extracts taken to dryness. The residue was dissolved in water and layered on a column of Sephadex Ga25 (2.5 cm x 25 cm). The column was eluted with water, collecting 5 m1 fractions. The fractions producing a green color also showed maximum absorption at 279 mp. These tubes were pooled, reduced in volume, and the extract subjected to preparative 24 thin-layer chromatography on a layer of cellulose using the upper phase of a n-butanol-acetic acid-water (4:125 v/v) mixture. The component was eluted from the layer and the eluent used for identifi- cation with commercial d-catechin (Calbiochem) used for comparison. IndoleaSaAcetic Acid Oxidase The seeds used for this study were taken from fruit which had been stored frozen. Protein determinations were according to the method of L0wry.2£.il' (53). Preparation of soluble enzyme fraction Forty grams of seeds taken from immature peach fruit which had been previously frozen were homogenized in 100 ml of 0.1 per cent Tween 80 solution in glass-distilled water. The detergent was used to eliminate formulation of protein-tannin complexes as described by Goldstein and Swain (24). The homogenate was centrifuged at 2000 x g at 4° for 20 minutes to remove cell wall debris. The supernatant was centrifuged at 22,000 x g for 30 minutes to give a mitochondria- free preparation. The preparation failed to show any IAA oxidase activity. The supernatant was freeze-dried, taken up in 2-3 ml of glass-distilled water, and placed on a column of Sephadex 6-25 (2.5 cm x 30 cm) packed in a column specially made for gel filtration (Pharmacia Fine Chemicals, Inc.). Elution of the column was accomplished with glass-distilled water. Three milliliter fractions were collected and their absorption at 280 mp read on the Beckman DK-2 Ratio Recording Spectrophotometer. Alternate tubes of the 25 first peak were checked for IAA oxidase activity. Tubes with enzyme activity were pooled and used for further studies. Enzyme assay Incubation mixtures were made up as follows: 1 ml of l x 10-3M MnCl2 (57), 1 ml of 1 x Io-3M 2,4-dich10rophenol (31), 0.1 ml of enzyme solution, 1 m1 of l x 10'3 IAA, and buffer to total 10 ml. The reaction was initiated by the addition of substrate. Incubations were carried out at 25°. The progress of the reaction was followed by measuring the change in the absorption spectrum of the reaction mixture as described by Ray (82), by following the change in absorption at 253 mp which measures oxindole formation (33), or by measuring IAA remaining using a colorimetric determination (76). For the latter, 4 m1 of a modified Salkowski reagent (3 ml of 1.5 M FeClg, 60 m1 of H2804, and 100 ml of H20) was added to 1 ml of the incubation mixture and the color read at 530 mu after 15 minutes on the Bausch and Lomb Spectronic 20. RESULTS RESULTS Auxin Identification Chemical Immature peach seeds collected during the years 1961 and 1962 were used in preliminary experiments to detect and identify indoles present in the tissue. In all seeds tested from these years, IAA was detected by paper chromatography using iSOpropanol- water as developing solvent (Table II). IAA was detected upon chromatography of the entire acid fractions. The use of isopropanol- water as developing solvent did not give the low mobility reported in the literature (67, 87). IAA could be detected upon chromatography of the entire acid fraction of the extract of 4 g of seeds. The neutral fraction obtained from the fractionation did not produce indole positive areas upon chromatography. This may have been a result of low concentrations of such auxins in the seed. The chemical detection of much lower levels of indole compounds in seed extracts was possible by thin-layer chromatography. The sensitivity range using thin-layer techniques approaches that for the bioassay. Extracts of seeds equivalent to 50 mg dry weight were routinely chromatographed on thin layers after partial purification of the extract by the cellulose column procedure. Three DMAC- positive spots were detected upon separation of extracts from seeds collected before, during, and after cytokinesis. The qualitative evidence for three indoles is represented by chromatographic separation of an extract of seeds collected during cytokinesis 27 28 Table II. Characteristics of Auxin Detected in Immature Peach Seeds Indole-B- Indole-B- Indole-3- Peach Characteristic acetic acid propionic acid butyric acid auxin RfIl (Paper) 0.74 —- -— 0.75 RfII (Paper) 0.33 -- -- 0.32 RfIII (TLc)2 0.43 0.52 0.58 0.43 RfIV (TLC) 0.52 0.57 0.59 0.55 RfV (TLC) 0.39 0.45 0.49 0.38 Color with DMAC reagent Violet Purple Purple Violet Color with FeClg; HC104 reagent Pink -- -- Pink Absorption 280, -- -- 280, maxima 288 mp 288 mp lThe solvents used for chromatography were: RfI - isopropanol, H20 (4:1); RfII - n-butanol, NH3, H20 (100:3:18); RfIII - chloroform, acetic acid (95:5); RfIV - chloroform, ethyl acetate, formic acid (50:40:10); Rfv - isopropanol, ethyl acetate, NH3 (45:35:20). 2TLC - Thin-layer chromatography on silica gel G or H. 29 (Table III).' Further experiments supported the identification of compound A as being IAA. In addition to a positive Chromogenic reaction, B was active in the straight-growth assay and has absorp- tion maxima typical for the indole nucleus, 280 and 288 mu. Re- chromatography and comparison of its mobility to ethyl indole-3- acetate, indole-S-acetonitrile, indole-B-acetaldehyde did not provide conclusive evidence as to the identification of B. Compound C did not move similarly to any known indole. The possible nature of these two unknowns will be presented in the discussion. Table III. Properties of DMAC-Positive Components in Seed Extract Straight-growth Compounds Rfl Color with DMAC activity Known IAA 0.43 Violet + A 0.42 Violet + B 0.57 Pink + C 0.87 Pink -- 1Chromatography on thin layer of silica gel G or H using chloroform-acetic acid (95:5 v/y). Evidence that compound A is IAA is summarized in Table II. The chromatographic results from the preliminary experiments are those represented for RfI. The other data represents typical results obtained upon rechromatography of the eluent from preparative scale separations of the extracts. The Rf values of known IAA in all five solvents agree well with those of the unknown acid. The 30 characteristic colors obtained with Chromogenic reagents and the ultraviolet absorption maxima also support the assignment of the auxin as being IAA. Bioassay The biological activity represented by the effect on growth 0f.éXEEE coleoptile sections by areas of the paper on which seed extracts were chromatographically separated offered a qualitative and quantitative means for detecting auxins in the seeds. The resulting auxin-like activity patterns of the chromatograms will be referred to as biograms. In the preliminary work, acid fractions were chromatographed on paper using isopropanol-water (4:1 v/%) as the developing solvent. A representative biogram from the separation of an extract from 50 mg of seeds collected at the time of cyto- kinesis is presented in figure 2. The resulting activity corres- ponded to the Rf of IAA, as well as the Rf's of other indolic auxins. Thus separation of the possible auxins was not possible with this solvent. 0n the basis of estimates of the quantities detected chemically, it was apparent that IAA was not the only auxin present. This supposition was supported after assaying sections of paper developed in butanol-ammonia-water. The biogramsobtained after separation of 50 mg dry weight equivalent extracts of seeds collected before, during, and after cytokinesis, respectively, are illustrated in figure 3. Authentic IAA has an Rf of 0.33 in the solvent system. The activity between 0.3 and 0:4 Rf is thus representative of IAA in the seeds. The presence of IAA in these extracts was confirmed by thin-layer 31 Figure 2 Biogram resulting from separation of extract of 50 mg of seeds collected on June 11, 1964, (cytokinesis) with isopropanol-water 32 0_ mo wd v0 N0 0 a _ _ 4 d d q _ _ _ 1 l ON on 029 on 00 NOIlVSNO‘IE 31lid03'100 WW NI 33 Figure 3 Biograms resulting from separation of'extracts of 50 mg of seeds collected (A) before, (B) during, and (C) after cytokinesis with butanol-ammonia-water. 1 q ‘ 1“ 34 =---------------—-------------------: _ — 4.0 )- A 3.0- 2.0- LOP _ _ _ A _ q q q . . .l - .1] . . P - Jtl _ _ _ _ IT- . LT. . . l] - ll . . — I1 Ilia - I . . lrl — 1' _ _ Ll. — lil- . _ L1 " 1.. _ . . B . C EL _ L _ p - F p 4L 0. o. o. o. o. 4 3 2 I 4 5:2 2. 20....40204w MJFQOMJOU |.O 0.8 0.6 0.4 ----—————-——-—-—----J_—— p... ————‘ 0.2 2.0- P—— LOP 35 chromatography. In each of the biograms, auxin activity also appeared between 0.8 and 1.0 Rf units. The lower level of activity in this region for sample C resulted from inhibitory concentrations evidenced by the unusual curling of the coleoptile sections. Possible identification of the auxins detected in this area will be presented later. Auxin activity between 0.1 and 0.2 Rf was also apparent in extracts of A and B. An inhibitor between 0.7 and 0.8 Rf increased in concentration with seed maturity. Thin-layer chromatography on silica gel H with 2-butanone- hexane (18:82 v/v) as developing solvent separates indoles which are non-acidic and leaves the acidic compounds at the origin (73). Extracts equivalent to 50 mg of seeds were chromatographed in this solvent system and the origins, which are minu5‘the'noneacidic auxins, were eluted and chromatographed on paper. The biograms of the acidic auxin fractions are shown in figure 4. The zones of activity at 0.1 to 0.2 Rf and 0.3 to 0.4 Rf resulted as before. In addition, activity was still observed at 0.8 to 1.0 Rf. Simple neutral indole auxins chromatograph in this area, but they would have been removed by the thin layer separation. The compound, which developed a pink color after spraying with DMAC reagent must thus be a new acidic auxin. No single auxin with these properties has been described in the literature. Identification of this acidic component apparently the same as compound C must await isolation of larger quantities. Chromatography of the original extracts on paper and development with hexand-chloroform-water (75:15:10 v/t, upper phase) 36 Figure 4 Biograms resulting from separation of extracts of 50 mg of seeds collected (A) before, (B) during, and (C) after cytokinesis after separation of non-acidic components. Method used is given in text. 37 A e A q q 4 a _ u q _ a dJ. a . . . . . . . . _ . - 1' h. IT- 1 - . _ . . . _ . . . . 1... 1.. . . . . . _ _ . 11 . 1r . . . . u . n 1 . 1| . T . _ _ . . . _ 1: _ . 11 . . _ _ . _ _ _ _ Jl _ .1! J _ _ . _ . . . — II. — Isl . . . . _ _ _ . . _ . 1.. . 1- _ . . _ . . n . r. . 11 , . .4 . . _ B . C _ _ _ p 4+ r _ _ _ p _ .1 _ p _ _ , r. _ 0. 0. 0. 0. 0. 0. 0. 0. 3 I. 3 2 I. 3 2 I. 5:2 2. 20....40204m m1..._.dom1_oo |.0 0.8 0.6 0.4 0.2 38 resulted in high activity at the origin (acidic components) and at the solvent front where ethyl indole-3-acetate, indole—B-aceto- nitrile, and indole-3-acetaldehyde also chromatographed. This was additional evidence that a neutral auxin was present, but as with the chemical eXperiments, identification was not possible. By using the straight—growth assay to measure the IAA on chromatograms of extracts, the auxin levels in the seeds before, during, and after cytokinesis could be compared as shown in figure 5. The time of cytokinesis was determined according to Leuty (48) as that period of growth at which the pericarp length reached 30 mm. The IAA level was highest during cytokinesis, which occurred in this case on June 11. Lombard and Mitchell (52) noted the increase of an acidic auxin, but were unable to demonstrate that it was IAA. These results show that IAA is present at an elevated level during cyto— kinesis. Catechin Identification Evidence that the compound in the acetone-water extracts was catechin (IV) is presented in Table IV. Catechin has been previously detected in mature peach fruit (7), but there was no previous evidence for its presence in immature seed tissue. 39 Figure 5 Comparison of IAA content to fruit growth and especially cytokinesis. Fruit growth was measured .by pericarp diameter and IAA content by bioassay of duplicate biogram sections of 0.3 to 0.4 Rf. 40 ("-1 WOHLNOO :10 1N3083d NI NOILVSNOWE 3'lLLd03'100 40 (—) WW '8313WVIO dHVOIHBd O O O O O 2: S2 E2 _ £2 ._ l I I I _ 0 )— —d .. a - 4 p- — " "l '- -1 '- '1 1— .\ -‘ 1- 0 — l l l L m to o 10 rd 8 N N — 16 20 24 l2 JUNE 3| MAY 41 Table IV. Evidence for Catechin in Immature Peach Seeds ‘ Effect on Color Absorption (mp) IAA Induced Compound Rfl with DMAC max. min. IAA Oxidase2 Growth3 Catechin 0.51 Green 278 252 Inhibits Synergistic Peach Seed Component 0.51 Green 279 252 Inhibits Synergistic 1Isopropanol-water (4:1 v/V) on paper. 2Measured spectrophotometrically using preparation from bean roots. 3Avena coleoptile straight-growth assay using 1 x 10-7 M IAA. It was noted that an aliquot of the purified preparation increased the growth-inducing effect of l x 10'7 M IAA in the Alena coleoptile straight—growth assay. This prompted the experiment shown in figure 6, which demonstrated the effect of catechin on IAA induced growth. Catechin at 1 x 10-3 M completely eliminated the induction of l x 10"6 M to l x 10'4 M IAA on growth and it appeared that some reduction of IAA induced growth at these concentrations prevailed at all concentrations of catechin. Levels of catechin less than 1 x 10'5 M increased growth induced by 1 x 10"7 M IAA. It has been previously reported (97) that l x 10"3 M catechin inhibited growth induced by 1 x 10-5 M IAA. . In view of a report by Letham and Bollard (47) that catechin retarded the growth of carrot tissue, it was of interest to note its effect on buckwheat root growth (figure 7). Inhibition of root 42 Figure 6 Effect of catechin on IAA induced growth of Avena coleoptiles. Molar concentrations of catechin are designated for the respective curves. 43 <4. LO ZO_._. 48 10' x sa10w ‘oaxoaisao WI o 00. 10 t N g I I I 1 I I 1 I 1 r- ; \ d ._ .\. q \. ._ ./ - ./ \. . 1— ./ ’.L ’././ ’I.” r- ,e ................ ., _ .‘ 1. i ______________ . ' ' ._ “.“1 1 \.~. _ f4 1' 1- .‘ 0 C 1 l l l I l I 1 I l 0. o N . 0Q ¢ 1—111111 082 it: Noudaossv l20 96 72 48 ML 0F ELUENT 24 49 Figure 9 Optimum pH conditions for IAA oxidase activity. IAA destroyed /ml was measured after 2, 4, and 6 hours. 50 0.2 - x $1.02 596580 3: 5.0 6.0 pH 4.0 51 Figure 10 Ultraviolet absorption spectra of reaction mixtures showing cofactor requirements for the peach IAA oxidase. Curve 1, complete reaction mixture after 1 minute; curve 2, same after 40 minutes; curve 3, same after 10 hours; curve 4, same minus 1 x 10‘4 M MnClz after 10 hours: and curve 5, same minus 1 x 10-4 M 2,4-dichlorophenol after 10 hours. 52 own 05 00m 0mm Com Ohm 1e £55633 com com OCN q d u - q q ..0 N6 nd to 0.0 00 5.0 NOILdHOSBV 53 Figure 11 Rate of 3-methy1eneoxindole formation catalyzed by peach IAA oxidase and its inhibition by catechin. The Lineweaver- Burke plot (inset) was prepared utilizing (1) no catechin (2) 2 x 10-7 M catechin, and (3) 4 x 10'? M catechin. AOD. AT 253 m): .0 m .0 A .0 N 54 _ N 01 o o o -IV(A0.D. AT 253 11.11/20 MIN.) 1 x 10'4(M0LES/L1TER) ’ O /./ TIME (HOURS) ,e e’.» 0 /°/ 0 55 almost completely disappeared after 2 hours in the absence of catechin as measured with the Salkowski reagent. Attempts to follow IAA destruction in the presence of catechin with the Salkowski reagent were unsuccessful. It appeared from the rate curve that catechin affected IAA oxidation in a non-competitive manner. The LineweavereBurke plot (12) in figure 11 substantiated the nonacompetitive manner of catechin inhibition. DISCUSSION DISCUSSION In View of reports by others (52, 87) the presence of free IAA in immature peach seeds was not unexpected. Evidence heretofore for the presence of IAA in the tissue-was accumulated from biological activity. The utilization of thin-layer chromatography combined with the bioassay made it possible to detect and identify low levels of the auxin. Even during cytokinesis, the level of IAA estimated on the basis of the limits of detection used, may not be greater than 4 or 5 mg per gram of dry weight. Stahly and Thompson (87) found from quantitative bioassays that the highest level of auxin in the seed of Halehaven peaches which they tentatively identified as IAA is 15 pg per gram dry weight. The levels of IAA for corresponding periods of development would not be expected to be consistent from year to year nor from cultivar to cultivar. L5W15.£E.il- (49) have found that the uncharacterized "citrus" auxin is also present in peach fruit and questioned whether the transient spots obtained by Stahly and Thompson (87) were really IAA. The experiments presented herein establishthe identification. No evidence was obtained in this work to suggest the presence of "citrus auxin" in the immature peach seed. The requirement for auxin in conjunction with a kinin for prdmoting cell division has been well established (58). Recently reported evidence has shown that kinin activity is abundant in the peach seed endosperm (78). One the basis of the increase in IAA at cytokinesis, it may well be required for the rapid cell division in endosperm; No data is yet available on concentrations of the kinin in these tissues relative to maturation of the fruit. 57 58 Since the IAA level apparently fluctuates, biochemical processes which may regulate its content should be considered. The data suggest that a system which regulates the rate of the oxidative destructiOn of IAA may be'functional for this purpose. The process would comprise the non-competitive inhibition by catechin of the IAA oxidase from peach seeds reported here. The non-competitive mode of inhibition suggests that the inhibitor is binding at a site removed from the active site of the enzyme (12). In the in 113:2 system, the substrate (IAA) is protected from oxidation by the presence of the inhibitor (catechin) and the rate of IAA destruction is dependent upon the level of the inhibitor. Chlorogenic acid, a competitive inhibitor of pea IAA oxidase (79), is also present in peach tissue and may also function as an inhibitor of the peach enzyme also. Alphysiological interrelationship between IAA, catechin, and IAA oxidase may be functional during maturation of the peach seed and fTUit. BiOIOqual analysis of the IAA content showed that it increased during cytokinesis and then decreased after completion of cell diVision. Donoho 33 El. (13) demonstrated that the rate of IAA destruction in 2322 was considerably less during cytokinesis. The present work'suggestS'that this fluctuation in oxidase activity may be manifested by the non-competitive inhibitory action of catechin. The rate of IAA'oxidase activity would thus be dependent on the amount of catechin bound to the enzyme at a particular time. When higher levels of IAA ate observed, higher quantities of catechin would be bound to the enzyme, invoking a decreased rate of oxidation. 59 Because of probable localization in the tissue and cells of both enzyme and inhibitor, this hypothesis could not be clarified in this study. The end-product of the in £3232 oxidation of IAA as catalyzed by the peach enzyme system was shown to be 3-methyleneoxindole. In view of recent observations (18) that this compound inhibits the growth of higher plants, considerations as to its effects on fruit growth and development would be in order. When IAA oxidase is most active, and therefore the IAA level is decreased, the concentration level of this inhibitory compound would be increased. The IAA oxidase system could thus be responsible for both a growth promoting compound (IAA) and a growth inhibiting compound (3—methyleneoxindole) and both may be physiologically important. In addition to IAA, other auxins were detected but not identified. One of these is non-acidic and is probably equivalent to auxin B mentioned earlier. Preliminary evidence suggests that it is either an ester of IAA, indole-B-acetonitrile, or indole-Boacetalde— hyde. The physiological function of auxin B is not evident at this time. It is possible that the acidic auxin (auxin C) which traveled to the higher Rf regions on paper in butanol—ammonia—water may be representative of a new class of auxins recently described as being present in the culture medium of excised tomato roots (89). The auxin from tomato roots was complex and included S—hydroxyindole— 3-acetic acid, 5-hydroxytrypt0phan and several amino acids. The complex auxin was soluble in ethyl acetate, had a high Rf value in 60 solvents containing ammonia, and gave a pink color with Ehrlich's reagent. The complex was reported to have activity in the straight- growth assay. Compounds which were equally complex were also detected in other plant roots. Although characterization of the peach seed component with similar properties has not been attempted, it is nevertheless tempting to speculate that the acidic auxin observed at the high Rf region by bioassay is perhaps a complex of the type described in tomato roots. If true, this is the first instance known to the author that a compound of this type has been described in immature peach seeds. No data is yet available which might explain a role for this complex in peach fruit maturation. SUMMARY SUMMARY An auxin, which was correlated by its relative concentration in the seed tissue with cytokinesis of the endosperm of the seed of immature peach fruit (Prunus persica, cv. Redhaven), was identified by its biological activity, paper and thin-layer chromatography, and ultraviolet absorption as indole-3-acetic acid. Evidence was obtained for the presence of another acidic auxin in extracts of the immature seeds. This compound had a Rf of 0.80—1.00 in butanol— ammoniaowater (100:3:18) and displayed typical auxin responses as well as colorimetric reactions typical of indoles. An indole-3- acetic acid oxidase system was detected and characterized in the soluble fraction of peach seed extracts. The enzyme had a pH optimum of 4.5 to 5.0 and required both Mn++ and 2,4-dichlorophenol for activity. The product of the reaction was shown to be 3-methy; leneoxindole. A polyphenol, catechin, was identified in the seed by chromatography. The compound was shown to be a non—competitive inhibitor of the oxidase enzyme. 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