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I ‘A 329;“ ‘ {Ht ‘ 'v -\ ,J This is to certify that the dissertation entitled THE IDENTIFICATION AND CHARACTERIZATION OF INDOLE-B-ACETYL-MYO-INOSITOL HYDROLASE FROM VEGETATIVETISSUE OF _Z_§_A MAYS L. presented by Prudence J. Hall has been accepted towards fulfillment of the requirements for A Ph.D. Botany & Plant Pathology degree in \Za TS Bahia; Major professor Date (I‘HJ\83 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 IV‘ESI_J RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from .—,—. your record. FINES will be charged if book is returned after the date stamped below. EM: Wrist!" (yawn new Ii” 1'. ,;‘ -.: a ~ ._ ;,, r“ 'tJ lwWfl \Enmwunlen». meg-u USE out? THE IDENTIFICATION AND CHARACTERIZATION OF INDOLE-3-ACETYL-fl19-INOSITOL HYDROLASE FROM VEGETATIVE TISSUE OF gEA MAYS L. By Prudence J. Hall 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 1983 ’D _/ —.} //' «j’g/ _ 5-? ABSTRACT THE IDENTIFICATION AND CHARACTERIZATION OF INDOLE-3-ACETYLffl19-INOSITOL HYDROLASE FROM VEGETATIVE TISSUE OF ZEA MAYS L. By Prudence J. Hall The presence of conjugates of the plant growth hormone indole-3- acetic acid (IAA) in vegetative tissue of plants in excess of the free acid is postulated to play a role in maintenance of endogenous hormone levels. In shoots of Zea mays L. seedlings, esters of IAA predominate and it is believed that these esters are enzymatically hydrolyzed to provide free IAA to the growing shoot. One ester has been isolated from corn shoots and was identified as indole-3-acetylgmyQ-inositol (IAInos). This dissertation describes the partial purification and characterization of an enzyme from corn shoots which hydrolyzes myo—inositol esters of IAA. The enzyme was partially purified by column chromatography using abaminohexyl agarose, hydroxylapatite, and Sephadex 6-100. In addition to the hydrolysis of IAInos, the enzyme preparation hydrolyzed the methyl esters of IAA and naphthyleneacetic acid, and the synthetic ester, a-naphthyl acetate. The rate of enzymatic hydrolysis was unaffected by added cofactors, sulfhydryl reagents, and metal cations, except for Mn2+which was somewhat inhibitory. Glycerol appeared to inhibit the appearance of free IAA, but other experiments demonstrated the possibility of a transesterification reaction. The hydrolysis of IAInos can be demonstrated in extracts from seeds and roots in addition to shoots. Additional data is presented Prudence J. Hall demonstrating the localization of enzymatic activity in the coleoptilar portion of the shoot. A calculation based on published data showed that the relative amount of esterified IAA in coleoptiles as opposed to mesocotyls parallels the distribution of enzymatic activty. IAA esterified to myg-inositol is present in substrate preparations as chemically and biologically resolvable isomers. Examination of substrate in reaction mixtures before and after enzymatic hydrolysis demonstrated that the chemically resolvable isomers disappeared at different rates. This leads to the suggestion that the endogenous rate of hydrolysis of IAInos is controlled in part by the abundance of some isomers relative to others. While the presence of hormone conjugates in plant tissues is recog- nized, and many studies have been published on their characterization and synthesis, the data presented in this dissertation constitutes the first report of an enzyme hydrolyzing a conjugate of IAA. To George T. Jones, Professor of Botany, Oberlin College and to Rich, Ethan, Eric and Kitty 11 ACKNOWLEDGEMENTS I wish to acknowledge the assistance of the members of my guidance committee, Norman Good, Derek Lamport and William Wells. I have appreciated their comments and advice. The guidance, encouragement and support of my major professor, Robert S. Bandurski, is gratefully acknowledged. It has been a priviledge to work in his laboratory. The interest and support of all those people in Dr. Bandurski's laboratory in the years I have been there - especially Aga Schulze, Jerry Cohen, Volker Magnus, Bill Pengelly and John Chisnell, must be acknowledged. Their friendship and their willingness to share their ideas have been most appreciated. I also acknowledge the support and encouragement of my husband and sons. They have encouraged me throughout my graduate studies and have helped out in every way. TABLE OF CONTENTS Page LIST OF TABLES...................................................... vi LIST OF FIGURES................................................ ..... vii LIST OF ABBREVIATIONS............................................... x GENERAL INTRODUCTION AND LITERATURE REVIEW.......................... 1 Conjugates of IAA: roles and metabolism......................... 3 Esterases: activities and identification in plants.............. 11 References 0.0.0.0...OOOOOOOOOOOOCOOOOOOOOOOOOOIOOOOOOOOOOOOO0.0. 17 EXPERIMENTAL I: PARTIAL PURIFICATION OF IAA-Mlg-INOSITOL HYDROLASE FROM ZEA MAYS L. VEGETATIVE TISSUECOOOOOOOOOOOOOOOOOOO0.0. 22 Abstract.O...OIOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOIOOOI. ....... O... 23 IntrOductionOC ..... .0.0.00.00.00.00.0IOOCOOOOOOOOOOOOOOOOOOOOOOOO 24 Mater1a15 and HethOdSOO0...0.0....0.00.00.0000...OOOOOOOOOOOOOOOO 26 Chemicals.0.0...O...0.0.0.0.0...0.0.0.0000...OOOOOOOOOOOOOOOOO 26 Preparation Of Plant Material0.00...0.0.0....OOOOOOOOOOOOOOOOO 26 BUfferSOOOCOOOOOO0.000.0.0000...0.00.00.00.00...OOOOOOOOOOOOOO 27 Protein Concentration......................................... 27 Polyacrylamide Gel Electrophoresis............................ 27 Esterase Activity Staining.................................... 27 Assay of IAInos Hydrolase Activity............................ 28 Assay of a-Naphthyl Acetate Esterase Activity................. 29 Results.......................................................... 30 Polyacrylamide Gel Electrophoresis............................ 33 Molecular Weight Estimates.................................... 39 HydrOIytic ActiVitieSOO0.0.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 39 Denaturation.................................................. 42 Other Sources of IAInos Hydrolase Activity.................... 42 DiSCUSSIOnOOOoooooooooooooooooooocoo-0000000000000000000000000... 45 ReferenCESOOOOOOOOOIOOOOOOOO0.0....0...00...0.0000000000000000.0. 49 EXPERIMENTAL II: CHARACTERIZATION OF IAA-MXQ-INOSITOL HYDROLASE AS ISOLATED FROM ZEA MAYS L. VEGETATIVE TISSUE ........... 52 AbStraCto00000000000oocoone...00000000000000.0000000000.00...coo. 53 IntrOductionOOOOOOOOOO0.0...0......0.0.0.0000...OOOOOOOOOOOOOOOOI 54 Materials and MethOdSOO00.0.0.0....0.0.0.00.0...OOOOOOOOOOOOOOOOO 56 ChemicaISIOOOOOOOOO00....OOOOOOOIOOOOOOOOOOOOO00.000.000.00... 56 BUfferSOOOOOOOOOO...00......OOOOOOOIOOOCOOOOOOIOOOOOOOOOOOOOCO 56 P1ant ExtraCtSooooooo000.000.0000.000000000000000000000000000. 56 iv Protein Concentrations........................................ IAInos Hydrolase Activity..................................... Hydrolysis of a-Naphthyl Acetate.............................. Preparation of Methyl Esters.................................. Hydrolysis of Methyl IAA (Me-IAA)............................. Hydrolysis of Methyl Esters of NAA, PAA and BA................ HPLC Resolution of IAInos Isomers and IAA..................... Results.......................................................... Characterization of IAInos Hydrolase.......................... pH optimum................................................. Tim course....0...OCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0...... Enzyme concentration....................................... Effect of additions to the reaction mixture................ Effect of added substrate.................................. Preferential hydrolysis of isomers......................... Esterolytic Activityes of IAInos Hydrolase and Esterase....... Hydrolysis of a-naphthyl acetate........................... Hydrolysis of Me-IAA....................................... Hydrolysis of other esters................................. Effects of glycerol........................................ Hydrolytic activities in coleoptiles as compared to mesocotyls.................................. Discussion.................................................... ReferenceSOC0.0.0.0000...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO CONCLUDING DISCUSSIONCOOOOOCOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOO0.... ReferenCESOO0..OOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0...0.00.00.00.00... APPENDIX A: pH Optima for HydrOIySESOOOO...OOOOOIOOOOOOOO...0...... pH optimum for 3H-IAInOS HYdr01y51SOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO pH Optimum for a-Naphthyl Acetate Hydrolysis..................... APPENDIX B: Affinity Chromatography...........u................... ReferencesCOOOOOO0.0.0.0000...O0.0.0.0....OOOOOOOOOOOOOOOOOOOOOOO APPENDIX C: Effects of NAD+ and NADP+ on Hydrolysis................ ReferenceSOO00.000.000.00...0..0......OOOOCOOOOIOOOOOOOOOOO0.0... APPENDIX D: Inhibition by Fluoride (F-) of a-Naphthyl Acetate HYdr01ySiSoooooococo-cocooooooooooooooooooooooo APPENDIX E: Environmental Perturbations and Enzyme Activities...... APPENDIX F: synthESiS Of Q'Naphthy] Acetate..OOOOOOIOOOOOOOOOOOOOOO Reference.’OOOOOCOOOOOOICC0.0.00......OOOOOOOOOOOOOOOOOO0.00...O. APPENDIX G: Calculations Used to Determine the Amount of IAA Produced by Hydrolysis of IAInos....................... APPENDIX H: Calculations Used to Determine the Amount of a-Naphthol Produced by Hydrolysis of a-Naphthyl Acetate........... LIST OF REFERENCESOOOOOOO0.000000000000000000000000..OOOOOOOOOOOOOOO 103 104 105 106 107 109 113 114 115 117 118 Table LIST OF TABLES Page EXPERIMENTAL I: PARTIAL PURIFICATION OF IAAjfllQ-INOSITOL HYDROLASE FROM ZEA.MAYS L. VEGETATIVE TISSUE Purification of IAInos hydrolase............................ 37 Purification of esterase.................................... 38 Comparison of enzymatic activities from crude extracts 0f 2’ mars.0.0.000...0000......OOOOOOOOOOIOOOOOOIO00.0...0.. 44 EXPERIMENTAL II: CHARACTERIZATION OF IAA-MlgleOSITOL HYDROLASE AS ISOLATED FROM Z§A_MAYS L. VEGETATIVE TISSE Effect of butyl alcohols on esterase activity............... 72 Comparison of enzymatic activities of coleoptiles and mesocotyls...0......0.00....OOOOOOOOOOOOOOOOOOOOOO0.......0. 74 Summary of properties of IAInos hydrolase................... 76 Comparison of properties of esterase and IAInos hydrolase... 78 CONCLUDING DISCUSSION Comparison of hydrolytic activities of esterase and IAInos hydrOIaseOOOOOO0.0.0.000...OOOOOOOOOOOOOOOOOO0..O..0. 91 Esterified IAA in whole shoots, coleoptiles and mesocotyISOOOOIOO0.0...OOOOOOOOOOOOOOOOOOOOOOOO00.0.00...00. 93 APPENDIX D Inhibition of fluoride of a-naphthyl acetate hydrolysis..... 108 APENDIX E Summary of results of environmental perturbations........... 110 vi LIST OF FIGURES Figure Page EXPERIMENTAL I: PARTIAL PURIFICATION OF IAA-M_YQ-INOSITOL HYDROLASE FROM g5 MAYS L. VEGETATIVE TISSUE 1 HydrOphobic interaction chromatography of Stage II enzyme. The elution pattern of enzymatic activities of Stage II enzyme from «raminohexyl agarose column (column size, 1.8 x 5.2 cm; flow rate 0.75 ml/min; fraction size, 10 ml/fraction) is shown. At fraction 10 protein bound to the column was eluted with 0.5M NaCl in Kphos buffer. Fractions were assayed for hydrolase (o) and esterase (0) activity as indicated, using 0.1 ml extract/assay. Fractions 2 to 4 were pooled for further purification....... 32 2 Gel filtration chromatography of Stage IV enzyme. The elution pattern of IAInos hydrolase activity of Stage IV enzyme from Sephadex G-100 (column size 1.6 x 33 cm; flow rate 10 ml/h 1 ml/fraction) is shown. Fractions were assayed for 3H-IAInos hydrolysis as indicated. Fractions 26-32 were pooled for further experiments......... 34 3 Gel filtration chromatography of Stage IVE enzyme. The elution pattern of Stage IVE esterase from Sephadex G-100 column (column size 1.5 x 23 cm, flow rate 10 ml/h, 1 ml/fraction) is shown. Fractions were assayed for a-naphthyl acetate hydrolysis as indicated. Fractions 10-25 were pooled for further experiments................... 35 4 Procedures for the partial purification of esterase and IAInos hydrolysis. This flow chart shows the procedures used to partially purify the esterase and IAInos hydrolase as described in the text.................................... 36 5 Gel electrophoresis of IAInos hydrolase and esterase. These tracings of polyacrylamide gel show the electro- phoretic separation of proteins present in partially purified IAInos hydrolase and esterase preparations. Lanes 1 and 4 were stained with Coomassie Blue. Lanes 2 and 3 were stained for a-naphthyl acetate esterase activity. The numbers indicate the distance of migration of a particular protein relative to the dye front (the bottom of the gel).......................................... 40 vii Figure Page Calibration of Sephadex G-100 gel filtration column. The relative retention volume represents the retention volumes of the standard proteins to the retention volume of Blue Dextran............................................. 41 EXPERIMENTAL II: CHARACTERIZATION OF IAAafllg-INOSITOL HYDROLASE AS ISOLATED FROM ZEA MAYS L. VEGETATIVE TISSE Summary of steps used in the partial purification of IAInos hydrOIaSEOOOOOOOOOOOOOOOOOOOOOOOOO0.00.0.0000...0.... 57 The effect of Snzyme concentration on the rate of hydrolysis of H-IAInos. Stage IV enzyme at 6 mg/ml was diluted with Kphos buffer and hydrolysis was assayed as described in the Methods section......................... 63 The effect of glycerol on 3H-IAInos hydrolysis. Fifty pl of Stage II enzyme (clarified 40 to 50% (NH4)7-SO4 fraction) was mixed with Kphos and glycerol to give the final concentrations of glycerol indicated in a total volume of 100 ul............................................ 64 Separation of isomers of IAInos. A. Chemically synthe- sized IAInos was chromatographed over a Partisil-IO ODS HPLC column. The flow rate was 2 ml/min, the solvent was ethanolzwatre (5:95). Fractions eluting under each of the 4 peaks were pooled, dried in vacuo, and resuspended in a small volume Of 50% aqueous 2-propanol. Aliquots from each were chromatographed in a TLC system using methylethyketone:ethyl acetate: ethanolzwater (3:5:1:1) as the solvent. B. TLC of standard IAInos and samples from HPLC. Lanes 1, 4, 6, standard IAInos; Lane 2, sample from peak 1; Lane 3, sample from peak 2; Lane 5, sample from peak 3; Lane 6, sample from peak 4.............. 66 Radioactivity associated with peaks of IAInos eluted from HPLC. Stage II enzyme (clarified 40 to 50% (NH4)ZSO4 fraction) was mixed with H-IAInos and the reaction mixture incubated 0 or 4 h. The reaction was terminated by the addition of an equal volume of 2-propanol. An aliquot was mixed with one ug chemically synthesized IAInos prepared in 50% aqueous 2-propanol, then chromato- graphed over a Partisil-IO ODS column. Fractions of 0.5 ml were collected and the radioactivity determined in each. The relative amounts of radioactivity associ- ated with each peak are shown in the upper portion of each panel.......................................... ........ 67 viii Figure Gel filtration chromatography of Stage IV enzyme. The sample was chromatographed over a calibrated Sephadex G-100 column (1.6 x 33 cm) in potassium phOSphate buffer. Fractions of one ml were collected. Aliquots of 100 pl were used for assays of 3H-IAInos hydrolase or a-naphthyl acetate esterase as indicated............................... Enzymatic hydrolysis of Me-NAA. Hydrolase or esterase preparations were incubated with 20 nmol Me-NAA. Control reactions contained no enzyme. An aliquot of each reaction mixture was chromatographed by HPLC (Partisil-IO ODS; ethanolzacetic acidzwater (40:1:59), one ml/min) to separate product from substrate. Me-NAA elutes at 15 ml while NAA elutes at 9 ml.................................... APPENDIX A Effect of pH on hydrolysis of 3H-IAInos. Aliquots of IAInos hydrolase were dialyzed against 0.05 M buffer at the pH indicated. Enzyme activity was measured as Page 70 71 descriDEd preViOUSIyCCOOC0.0...O...OOOOOOOOOOOOOOOOOOOOOOOO. 101 Effect of pH on hydrolysis of a-naphthyl acetate. Aliquots of Stage I enzyme were suspended in 0.05 M buffer at the pH indicated. Activity was measured as described prev10u51y00000......OOOOOOOOOOOOOOOOOOIOOO0.0.... 102 ix BA BSA 0 DEAE EDTA GLC HPLC IAA IAInos Kphos Me-BA Me-IAA Me-NAA Me-PAA NAA PAA TLC Tris LIST OF ABBREVIATIONS benzoic acid bovine serum albumin Daltons diethylaminoethyl ethylenediaminetetraacetic acid gas liquid chromatography high performance liquid chromatography indole-3-acetic acid indole-B-acetylgmyg—inositol potassium phosphate buffer benzoic acid, methyl ester indole-3-acetic acid, methyl ester naphthylene acetic acid, methyl ester phenylacetic acid, methyl ester naphthyleneacetic acid phenylacetic acid thin layer chromatography Tris (tris(hydroxymethyl)aminomethane) chloride buffer GENERAL INTRODUCTION AND LITERATURE REVIEW Indole-B-acetic acid (IAA) is a growth regulating hormone in plants. Many higher plants, during early non-photosynthetic growth, may be considered to be limited in their growth by IAA since removing the plant's source Of IAA causes a cessation of growth while supplying additional IAA increases the rate Of growth (62). Curiously, plants in which growth is limited by IAA (in the sense that exogenous IAA will increase the rate of growth) contain large amounts of IAA, but most of the IAA is in a covalently conjugated, and apparently, inactive form (4). In this situation the rate Of conjugate hydrolysis may well determine the rate of plant growth. Most of the IAA in seedlings of Zea mays is present as esterified IAA, some is present as myg-inositol esters. The corn seedling thus provides an Opportunity to study the enzymology of hydrolysis of an IAA conjugate. The purpose of this dissertation is to describe the enzyme which hydrolyzes the myo-inositol esters of IAA in vegetative tissue of z. mays. Three projects in the laboratory of Robert S. Bandurski made this work possible. First, Lech Michalczuk demonstrated the synthesis of indolyl-3-acetylfimygginositol (IAInos) by enzymes in the endosperm Of developing ;. gays kernels (40). Michalczuk and John Chisnell (42) extended this work by biologically synthesizing radioactive IAInos in quantities sufficient for use in other experiments. Second, Janusz Nowacki and Jerry Cohen chemically synthesized IAInos (45). Third, John Chisnell identified IAInos as an endogenous IAA ester in shoots of Z. mays (12). At the outset of this work the overriding questions were: 1) Can enzymatic hydrolysis of IAInos be demonstrated in extracts from ;. gays vegetative tissue? 2) Will this enzyme hydrolyze other ester substrates, most notably other IAA esters, esters of IAA analogues, and artificial esters? 3) Is this enzyme an esterase? If so, will any esterase be able to hydrolyze IAInos? Is there only one enzyme which hydrolyzes IAInos? 4) Is this enzymatic activity localized within the plant? 5) What controls the activity of the enzyme? Can we observe changes in activity of the enzyme in response to perturbations in the environment? The roles of endogenous and exogenous esters of IAA in plant tissues are first discussed followed by a brief discussion of esterases - why plant biologists have been interested in them, and some things that have been learned about them. The main body of the dissertation is composed of two papers to be submitted for publication: (1) Partial Purification of IAAgmyQ-inositol Hydrolase from lea mays L. Vegetative Tissue, and (2) Characterization of IAAjmyo-inositol Hydrolase as Isolated from Zea .mays L. Vegetative Tissue. A discussion section follows in which some problems encountered during the course of these studies are raised, and some suggestions for their solution are offered. Results of experiments are discussed and interpreted with respect to the questions asked at the outset. Appendices are included which 1) report the results of experi- ments too incomplete to be included in the main text, and 2) provide detailed experimental procedures for the benefit of others interested in continuing these studies. Conjugates of IAA: roles and metabolism Why are plant physiologists interested in hydrolysis of conjugates of IAA? The chemical identification of IAA as an auxin was published by Kogl gt al in 1934 (36). The source of the IAA was urine, but this "heteroauxin" was active in promoting growth in plant tissues. “Heteroauxin” was isolated by Thimann (59) from Rhizopus suinus and it too was chemically identified as IAA. At about the same time several investigators, in the interests of increasing yields of IAA, tried different methods for extracting auxins from plant tissues. Alkali extraction was found to increase yields of auxin as much as 20 fold. In 1941 Hatcher and Gregory (32) reported that a maximal amount of auxin could be extracted with water or by diffusion into agar from develOping grains of rye 5 to 6 weeks after anthesis, and that no auxin activity could be extracted from the dry grain. The same year Avery gt 31 (1) published an extensive report concerning the increased amount of auxin that could be extracted from corn using water at pH 9 to 10. These workers (1) made the distinction between free auxin and an auxin precursor and speculated about the nature of the auxin precursor. The ease of releasing "free" auxin from the precursor by alkaline hydrolysis suggested to them that the precursor was an ester. They hesitated, however, to definitely class it as such because the precursor was insoluble in several organic solvents that a simple ester of IAA should be soluble in, it was inactive in growth tests whereas the methyl and ethyl esters of IAA were active. They were relatively certain it was not an amide. The success of Avery and co-workers in using alkaline extraction, however, prompted Hatcher to repeat his experiments. In 1943 Hatcher (31) reported that an inactive auxin derivative continued to accumulate in developing grains of rye even after free auxin was no longer detectable, and that alkaline extraction yielded up to 20 times as much active auxin as the grain ripened. Meanwhile Haagen-Smit (27) established that the auxin released from a precursor in corn kernels was indeed IAA. He characterized the IAA primarily on the basis of its melting point and the fact that there was no change in the melting point when the IAA was mixed with authentic IAA. Berger and Avery (8) confirmed the identity of the auxin released from the precursor as IAA. At that time the primary method used to determine whether auxin was present was by bioassay. In 1935 Cholodny (13) described experiments in which he used sections of endosperm from Aléflé kernels to induce negative curvature in Alena coleoptiles, that is, the coleoptiles grew faster on the side to which the block of endosperm was applied. He sliced dry, unimbibed endosperm, moistened it and placed it on the side of a decapitated, etiolated AXEEE coleOptile. A negative curvature of 30 to 40° develOped in 1.5 h at 20 to 21°C. 0n the basis of two experiments Cholodny ruled out the possibility that free auxin present in the endosperm caused the curvature. First, he showed that dry seeds lost growth promoting ability when heated to 100°C. He knew that auxin was heat stable, so he concluded that whatever growth promoter was present in the endosperm was not auxin in its usual form. Second, he showed that no growth promoting substance was extracted by 96% ethanol, as auxin would be. However, if seeds were soaked for 12 h, then extracted with water, auxin activity was found in the water extract. Furthermore, the substance in the water was alcohol and ether soluble, as auxin would be. So seeds which had begun to germinate could produce the growth promoting substance. Germination was not required, however, as seeds in which the embryo had been killed could still induce curvature. His basic observations were that some compound in the endosperm could induce curvature if moistened. The compound was not itself auxin, but auxin seemed to come from it. Cholodny concluded that the 51222 endosperm hormone is liberated from a precursor by hydrolyzing enzymes (13, p. 298). He further suggested that since the endosperm was a rich source of auxin precursor, that it should be possible to determine the structure of the growth hormone (by chemical means) becuase there was so much of it in the endosperm (13, p. 306). Although Cholodny's work did not receive the attention it deserved at the time (19), Cholodny did set the stage for later studies of the hydrolysis of conjugates of IAA and the role of conjugates in hormone physiology. The realization that seeds contained large quantities of a precursor of this important hormone was to have repercussions in the study of IAA effects for years. Interest in esters of auxins and auxin analogs had already emerged in the mid-1930's. Zimmerman and his associates (63) at the Boyce Thompson Institute synthesized a series of esters of IAA, naphthalene acetic acid (NAA), and phenylacetic acid (PAA) among others, and studied their effectiveness when applied in lanolin in causing bending in tomato shoots and root initiation in Kalanchoe and tomato. They also studied aerial roots in Eiééflé where auxin applied in the region of elongation would inhibit elongation, and promote bending and branching. The roots would bend towards the side of application which contrasted with the direction of bending observed in shoots. They studied the effects of auxins and their synthetic esters on tomato plants by injection into the stem or by uptake from the soil. The methyl and ethyl esters of IAA were consistently more effective in all test systems as compared to other auxin esters, and methyl IAA was more effective than ethyl IAA, which was in turn more effective than butyl IAA. The methyl esters of NAA and PAA were slightly less effective than their respective free acids. Avery gt .al (2) studied synthetic auxins using the Aygng curvature assay. They concluded that IAA and indolebutyric acid and their derivatives, the methyl esters and the potassium salts, were more active than other analogs tested. As shown later by Sell gt El (57) the methyl and ethyl esters of IAA were 100 times more effective than free IAA in stimulating parthenocarpy in tomatoes. The search for endogenous esterified auxins began shortly thereafter (8,9) and continues until the present (12,33,49) although a catalog of IAA esters has been achieved only for 2. may; endosperm. After Hamilton gt 31 (28) demonstrated that half of the esterified IAA was insoluble and the other half soluble in water, a series of papers by Bandurski and coworkers appeared which elegantly identified and characterized the water-soluble low molecular weight esters. Labarca (39) identified the IAInos and IAInos-arabinoside esters. Ueda (61) added IAInos-galactose to the list. Ehmann (20,21) studied IAA-glucose and di- and tri-IAInos. At the present time our understanding of how the plant uses its con- jugated auxin is incomplete, although the importance of its role is very apparent (3,4,5,14). From studies of esterified IAA in ;, may; endosperm and shoot tissue, the notion of conjugates of IAA playing a major role in a homeostatic mechanism for control of hormonal levels emerged (3,4,5,37). This hypothesis proposes that IAA conjugates are formed and released in plant tissue as a way to control the levels of free hormone. The hypothesis rests in part on the assumption that the level of free hormone for a particular growth state is critical to the plant for integrating growth and development. This control is thought to work in the following way: Degraded IAA esterification Transported IAAe—IAAF=====§IAA-x hydrolysis IAA-target (hormone utilization) Degraded IAA A steady state concentration of IAA is maintained in one part of the plant that is appropriate to the environment of that particular part. A change in the environment requiring an alteration in growth results in resetting the homeostatic mechanism such that a new appropriate level of IAA concentration is achieved. The major precursor of free hormone is the pool of conjugated hormone. To remove hormone from the pool of free hormone four routes are envisioned: a conjugate may be synthesized; the hormone may be degraded; the hormone may be transported out of the tissue; or the hormone may be used in growth promotion. A major cornerstone of the hypothesis is the observation that all plant tissues so far examined contain IAA in the conjugated state in excess of that in the free state. Bandurski and Schulze (6) examined the free and bound IAA of a number of different plant tissues and found no tissue which had more free IAA than bound. It also appeared that, except for Aygna vegetative tissue in which an amide conjugate predominates, esterified IAA was the predominant bound form in the cereal grain plants. Another important observation contributing to the hypothesis is that of Ueda and Bandurski (60). They measured the disappearance of IAA conjugates in germinating Z:.EEX§ kernels. The amount of free plus ester IAA recovered after alkaline hydrolysis of entire plants decreased during the first 96 h of germination from nearly 70 mg IAA/kg dry weight to about 7 mg/kg. The disappearance had also been studied by Corcuera (15). The use of the auxin reserves of the seed during germination again emphasized their importance in hormone balances. Utilization of seed auxin reserves also implies that a mechanism exists for releasing free hormone from conjugates. The most obvious way for hydrolysis to occur in viyg is by enzymatic hydrolysis. In fact Kogl £3.21 (36) had employed biological digestion as a way to obtain large l (27) did similar experiments and found amounts of IAA. Haagen-Smit gt that they could obtain 6.9 mg IAA/kg wheat (dry kernels) as compared to 0.2 mg/kg by water extraction. Chymotrypsin at pH 7 only liberated 0.76 mg/kg. Although enzymatic hydrolysis of IAA esters is implicated in the endosperm, the homeostatic hypothesis assumes operation of the homeostatic mechanism in all parts of the plant. One system which readily lends itself to study of the homeostatic mechanism is the shoot of a dark grown ;, mgys seedling. The shoot responds to environmental stimuli by altering its growth pattern. For example, a seedling laid on its side will grow so that the shoot bends and eventually grows vertically again (62). Or, a shoot exposed to a brief flash of bright light grows at a reduced rate (22). Two observations about hormone levels in shoot tissue greatly enhance the appeal of homeostasis as an explanation. First, IAA conjugates are present in vegetative tissues of the 4 day corn shoot (6,48) and second, the ratio of free IAA to esterified IAA changes in response to an environmental stimulus - photoinhibition of growth causes a reduction in free IAA and an increase in esterified IAA (7). The enzymatic formation of esterified IAA in developing ;. may; kernels has been studied extensively and the enzymes synthesizing all the low molecular weight esters have been described. Michalczuk (40,41) showed that IAA—B-D-glucose is synthesized from IAA and UDP-glucose by one enzyme. A second enzyme catalyzes the acylation of myg-inositol resulting in the products IAInos and glucose. Corcuera st 31 (17) studied the enzymatic synthesis of IAInos-galactose from IAInos and UDP-galactose. Corcuera and Bandurski (16) then described the two step synthesis of IAInos arabinose from IAInos and UDP-xylose, presumably involving a 4'-epimerase. These reactions account for the synthesis of all the known low molecular weight esters of IAA in ;. mays. 10 The use of IAA esters by plants has also been studied. Besides studies described above, Seeley gt_al_(56) used paper chromatography as a tool to demonstrate the hydrolysis of exogenous synthetic IAA esters. They knew that Me-IAA was active in the A3222 extension assay and in the pea curvature assay. They floated segments of wheat shoots in solutions containing Me-IAA, then concentrated the solution and chromatographed an aliquot using n-butanol:ammonia:water as the solvent system. Two Ehrlich positive spots were observed, one at the Rf of Me—IAA, the other at the Rf of IAA. Other plants yielded different products. For instance, peas appeared to metabolize Me-IAA to IAA and indoleacetonitrile. IAA amide conjugates have been shown to be active in several plant tissue culture systems by Hangarter gt_al (30). They felt the conjugates served as slow release forms of the hormone. Hangarter and Good (29) demonstrated metabolism of the conjugates by applying [1-14CJIAA-L-alanine and [1-14CJIAA-glycine to pea stem segments. IAA was presumably hydrolyzed from the conjugate, and they measured the release of 14C02 resulting from decarboxylation of IAA. Since the D-isomers of amino acids were not active, the decarboxylation was assumed to be enzymatic. Hydrolysis of IAInos in the shoot was considered by Nowacki and Bandurski (44) in their study of transport of IAInos. If they applied 14C-IAInos to the cut endosperm of a 4 day ;. gays seedling, they could reisolate radioactivity from the shoot. 0f the radioactivity appearing in the shoot, 7% was free IAA, 56% was IAInos, 29% was in esters other than IAInos, and 8% was unaccounted for. The question was whether the 14C-IAA recovered from the shoot arose from the hydrolysis of 14C-IAInos in the endosperm or in the shoot. They knew how much 14C-IAInos had been J1 applied to the endosperm, the pool size of IAInos and IAA, and the rate of IAInos hydrolysis in the endosperm. They reasoned that since nearly all of the IAA, free and esterified, occurs in the endosperm, that if hydrolysis of the 14C-IAInos occurred in the endosperm, the resulting 14C-IAA would be greatly diluted by endogenous IAA, and consequently any 14C-IAA which was transported from the endosperm to the shoot would be virtually undetectable. They concluded that since they could detect labeled IAA in the shoot, hydrolysis of IAInos must have occurred in the shoot. Esterases: activities and identification in plants If the hydrolysis of IAInos is enzymatically catalyzed, what kind of enzyme might be expected to catalyze the reaction? Because the substrate is an ester of a carboxylic acid, catalysis by a carboxylic acid esterase (EC 3.1.1) is likely. In general, the hydrolytic enzymes (EC 3) catalyze a nucleophilic diSplacement reaction in which a nucleOphile forms a bond with an electron deficient carbon (or phosphorus) atom, diSplacing some other atom, such as oxygen, nitrogen or sulfur. The leaving group is the displaced atom plus its bonding electrons and anything else attached. When the electron deficient carbon is a carbonyl and the displaced atom is an oxygen, then the enzyme is an esterase. The reaction for ester hydrolysis can be diagrammed as follows: 9 ’g-OR' + ‘OH" + H+--) HO- C‘ + R'OH R R 12 The animal carboxyester hydrolases have been subdivided into various categories (38). There is at least some agreement that esterases might be subdivided on the basis of their catalytic activity in the presence of different organophosphorus inhibitors. The A-esterases hydrolyze aromatic esters and are not inhibited by diethyl penitrophenyl phosphate (Paraoxon) or diisopropylfluorophosphate (DFP). In fact, an A-esterase will hydrolyze Paraoxon to give p-nitrophenol and a diethyl phosphate anion. Many A-esterases are also stimulated by Ca2+ and are inhibited by chelating agents (43). The B-esterases or serine hydrolases have also been called the non-specific esterases. They are stoichiometrically inhibited by organophosphorus compounds without hydrolyzing them. The role of serine at the active site of serine hydrolases allows a wide variety of enzymes to be classified as serine hydrolases. These enzymes include cholinesterases and Chymotrypsin. Esterases enjoy a wide distribution in animals and plants. Esterases have been reported in citrus fruits, carrots, radish seedlings, nearly all cereal grains, etc. One common way to establish the presence of esterases in a plant is to electrOphoretically fractionate a crude extract in a starch gel, then assay the enzymatic activity using a simple ester such as a-naphthyl acetate as the substrate. If the assay is done in the presence of a diazonium salt, such as Fast Violet B, then the product of hydrolysis a-naphthol, reacts with the salt to form an azo compound at the site of the reaction, and a colored band results. In crude plant extracts the number of esterase bands can be overwhelming: One early investigation of esterase activity in plants was reported by Jansen gt al (35) who characterized a citrus acetylesterase with respect to several substrates. Schwartz, in 1960 (55), picked endosperm 13 of developing maize kernels as the system in which he could study mutant genes and their transmission by looking at the gene products. In addition to providing a material for genetic analysis which had a short generation time, in which mating could be controlled, and in which large numbers of progeny could be studied, maize was considered excellent for cytogenetic analysis. Schwartz used starch gel electrophoresis to separate esterases from crude (he squeezed the kernels with a pliers, then collected the exudate on filter paper wicks to apply to the gel) extracts. He stained for esterases using a-naphthyl acetate as the substrate and visualized the bands by coupling with Fast Blue RR. His findings opened up a new area of interest for plant biologists. His corn lines were highly inbred and well defined. Three forms of the enzyme could be found in his parental stocks - they were defined on the basis of migration rates towards the cathode. From crossing studies he found hybrids which showed both parental types of esterase plus a hybrid esterase which was intermediate in its migration rate. Not only had he devised a simple method to assay gene products, but he had also demonstrated some potentially interesting features about inheritance of specific gene products in plants. Although he said there was "no easily detectable phenotype alteration associated with these mutant genes" (55, p. 1212), he suggested the possibility that the formation of hybrid enzymes could be a factor in hybrid vigor. The study of plant isozymes exploded (51,52,53). Esterase isozyme variants are still being studied today (54; Q. Yi, personal communication). Unlike animal esterase isozyme studies, those in plants, that I am aware of, have not really added to any understanding of the role of esterases in growth and development. Although esterase isozyme studies in animal tissues also 14 concentrate on breeding and transmission of hybrid traits, some functions of the enzymes are known (11,25). Not only did geneticists and plant breeders study esterases, but so did plant histochemists. For example, Gahan and McLean (24) wanted to determine the subcellular localization of esterases in Vicia faba root tips. Biochemical studies had shown that most hydrolytic activities were associated with the vaculoles in plant cells, but cytochemical studies had shown two locations for esterases, one in association with acid phosphatases, the other in particles adjacent to the cell wall. Gahan and McLean reported that esterase activity was located at several places intracellularly. They could only guess at the roles esterases might be playing. There are a few biochemical studies on non-specific esterases from plant cells (10,34) in which the esterases are characterized with respect to different artificial substrates. Studies in which an esterase is characterized with respect to an endogenous substrate include that by Jaffe and coworkers who published a series of papers in which they described the isolation and characteriza- tion of cholinesterases from mung bean roots (23,50). Goodenough and Entwistle (26) isolated an esterase from Malus pumila fruit. The enzyme, purified to homogeneity, hydrolyzed a series of endogenous volatile esters. These esters had been observed to increase as fruits ripened and were thought to "enhance the appeal of the fruit to animals likely to eat it and thus distribute the seeds" (26, p. 145). The specific activity of the enzyme increased as the fruit increased in size. Two papers (46,58) from Strack and coworkers are of special interest in studies of plant esterases. These workers looked at the hydrolysis of sinapine (sinapoylcholine), a naturally occurring ester in seeds of most 15 Brassicaceae. Sinapine is degraded during germination but it was not known if the degradation was catalyzed by non—specific esterases or by an esterase specific for sinapine. Strack gt 91 (58) isolated enzyme activity from cotyledons of Raphanus sativus which turned out to be quite specific for sinapine, and was only weakly inhibited by the organophosphorus serine esterase inhibitors. Neostigmine, which Riov and Jaffe (53) reported to be specific for plant cholinesterases was also only weakly inhibitory. Strack concluded that sinapine esterase is not a cholinesterase and is not related to cholinesterase from Phaseolus aureus as described by Jaffe. Enzyme activity was localized in the cotyledons and increased during early germination. Esterase activity hydrolyzing indophenyl acetate was maximal in dry seeds; only trace amounts were hydrolyzed at 48 hr when sinapine hydrolysis was maximal. Zymograms of cotyledon extracts at various stages of germination showed several bands stained for a-naphthyl acetate esterase activity. None of these bands corresponded in location to the single band which stained for sinapine esterase activity. The implication of Strack's experiments is that studying esterolytic activity may be far from straightforward. To further compound things, some other well-characterized enzymes also have esterolytic activity. Perhaps it is not too surprising that some proteolytic enzymes will hydrolyze esters of amino acids. For example, a protease from Staphylococcus aureus (18) hydrolyzes the phenyl ester of a peptide substrate. A better studied example of a multi-functional enzyme is 3-phosphoglyceraldehyde dehydrogenase. Park gt a1 (47) described the hydrolysis of p-nitrophenyl acetate by crystalline NAD-free enzyme. The 16 enzyme also catalyzed the oxidation of acetaldehyde to acetyl phosphate, the transfer of the acetyl group to inorganic phosphate, arsenate, Coenzyme A and other sulfhydryl acceptors, and the hydrolysis of acetyl phosphate to acetate and inorganic phOSphate. A study of the mechanism of the latter reaction led to their study of esterase activity. The literature reviewed in this section was selected for the purpose of laying the ground work for consideration of the experiments to be described in the next section. I hope I have convinced readers that an attempt to demonstrate the hydrolysis of IAInos was a worthwhile undertaking. Although its characteristics may, in the end, make it an atypical IAA ester, its existence in the shoots of corn seedlings is acknowledged. The two related questions - can any esterase hydrolyze IAInos (or, are there any that don't?) and, is IAInos hydrolase an esterase? - seem appropriate in view of what is known about esterases. 10. 11. 12. REFERENCES Avery, GS, Jr, J Berger, B Shalucha 1941 The total extraction of free auxin and auxin precursor from plant tissue. Amer J Bot 28: 596-607 Avery, GS, Jr, PR Burkholder, HB Creighton 1937 Avena coleoptile curvature in relation to different concentrations of certain synthetic substances. Amer J Bot 24: 226-232 Bandurski, RS 1978 Chemistry and physiology of myg-inositol esters of indole-3-acetic acid Ln WW Wells, F Eisenberg, eds, Cyclitols and the Phosphoinositides. 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Amer J Bot 31: 203-208 Carino, LA, MW Montgomery 1968 Identification of some soluble esterases of the carrot (Daucus carota L.). Phytochem 7:1483-1490 Chao, J 1983 Purification and characterization of rat urinary esterase A, a plasminogen activator. J Biol Chem 258: 4434-4439 Chisnell, JR, RS Bandurski 1982 Isolation and characterization of indol-31yl-acetyl-myQ-inositol from vegetative tissue of Zea may . Plant Physiol 69: $55 17 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 18 Cholodny, N 1935 Uber das Keimungshormon von Gramineen. Planta 23: 289-312 Cohen, JD, RS Bandurski 1982 Chemistry and physiology of the bound auxins. Annu Rev Plant Physiol 33:403-430 Corcuera, LJ 1967 Estudio de indolacetilinositoles en granos de maiz. BSc thesis. Universidad Catolica de Chile, Santiago Corcuera, LJ, RS Bandurski 1982 Biosynthesis of indol-3-yl-acetyl- .myg-inositol arabinoside in kernels of gga_mays L. Plant Physiol 70: 1664-1666 Corcuera, LJ, L Michalczuk, RS Bandurski 1982 Enzymic synthesis of indol-3-ylacetyl-myg-inositol galactoside. Biochem J 207: 283-290 Drapeau GR 1976 Protease from Staphylococcus aureus. In_L Lorand, ed, Methods in Enzymology, Vol 45. Academic Press, New York, pp 469-475 Ehmann, A 1973 Indole compounds in seeds of leg may . PhD thesis. Michigan State University, East Lansing Ehmann, A 1974 Identification of 2-0-(indole-3-acetyl)-D- glucopyranose, 4-0-(indole-3-acetyl)-D-glucopyranose and 6-0-(indole-3-acetyl)-D-glucopyranose from kernels of Zea ma 5 by gas-liquid chromatography-mass spectrometry. Carbohydr Res g4: 99-114 Ehmann, A, RS Bandurski 1974 The isolation of di-O-(indole-3- acetyl)-myg-inositol and tri-O-(indole-B-acetyl)-myg-inositol from mature kernels of Zea may . Carbohydr Res 36: 1-12 Elliot, WM, J Shen-Miller 1976 Similarity in dose responses, action spectra and red light responses between phototrOpism and photoinhibition of growth. Phytochem Photobiol 23:195-199 Fluck, RA, MJ Jaffe 1974 Cholinesterases from plant tissues. V. Cholinesterase is not pectin esterase. Plant Physiol 54: 797-798 Gahan, PB, J McLean 1969 Subcellular localization and possible functions of acid e-glycerophosphatase and naphthol esterases in plant cells. Planta 89: 126-135 Gilbert, 0G, RC Richmond 1982 Esterase 6 in Drosophila melanogaster: reproductive function of active and null males at low temperature Proc Natl Acad Sci 79: 2962-2966 Goodenough, PW, TG Entwistle 1982 The hydrodynamic properties and kinetic constants with natural substrates of the esterase from Malus pumila fruit. Eur J Biochem 127: 145-149 Haagen-Smit, AJ, W0 Leech, WR Bergren 1942 The estimation, isolation, and identification of auxins in plant materials. Amer J Bot 29: 500-506 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 19 Hamilton, RH, RS Bandurski, BH Grigsby 1961 Isolation of indole-3-acetic acid from corn kernels and etiolated seedlings. Plant Physiol 36: 354-359 Hangarter, RP, NE Good 1981 Evidence that IAA conjugates are slow-release sources of free IAA in plant tissues. Plant Physiol 68: 1424-1427 Hangarter, RP, MD Peterson, NE Good 1980 Biological activities of indoleacetylamino acids and their use as auxins in tissue culture. Plant Physiol 65: 761-767 Hatcher, ESJ 1943 Auxin production during development of the grain in cereals. Nature 151: 278-279 Hatcher, ESJ, FG Gregory 1941 Auxin production during the development of the grain of cereals. Nature 148:626 Hinsvark, 0N, WH Houff, SH Wittwer, HM Sell 1954 The extraction and colorimetric estimation of indole-3-acetic acid and its esters in developing corn kernels. Plant Physiol 29: 107-108 James, DJ, ARW Smith 1974 A biochemical study of non-specific esterases from plant cells, employing the histochemical substrate, naphthol AS-D acetate. Histochem J 6:7-23 Jansen, EF, R Jang, LR MacDonnell 1947 Citrus acetylesterase. Arch Biochem Biophys 15: 415-431 Kogl, F, AJ Haagen-Smit, H Erxleben 1934 Uber ein neues auxin ("Heteroauxin") aus Horn. Z Physiol Chem 228: 90-103 Kopcewicz, J, A Ehmann, RS Bandurski 1974 Enzymatic esterification of indole-3-acetic acid to myQ-inositol and glucose. Plant Physiol 54: 846-851 Krisch, K 1971 Carboxylic ester hydrolases. In_PD Boyer, ed, The Enzymes, Ed 3, Vol 5. Academic Press, New York, pp 43-69 Labarca, C, PB Nicholls, RS Bandurski 1965 A partial characterization of indoleacetylinositols from Zea may . Biochem Biophys Res Comm 20: 641-646 Michalczuk, L, RS Bandurski 1980 UDP-Glucosezindoleacetic acid glucosyl transferase and indoleacetyl-glucose:myQ-inositol indoleacetyl transferase. Biochem Biophys Res Comm 93: 588-592 Michalczuk, L, RS Bandurski 1982 Enzymic synthesis of 1-0-indol-3- ylacetyl-B-D-glucose and indol-3-ylacetyl-myQ-inositol. Biochem J 207: 273-281 Mighalczuk, L, JR Chisnell 1982 Enzymatic synthesis of 5- H-in ole-3-acetic acid and 5-3H-indole-3-acetyl-myg-inositol from 5- H-L-tryptophan. J Labelled Comp Radiopharm 19: 121-128 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 20 Myers, DK 1960 Carboxyl ester cleavage. .13 P0 Boyer, H Lardy, K Myrback, eds, The Enzymes, Ed 2, Vol 4. Academic Press, New York, pp 475-483 Nowacki, J, RS Bandurski 1980 M o-inositol esters of indole-3-acetic acid as seed auxin precursors of Zea mays L. Plant Physiol 65: 422-427 Ngwacki, J, J0 Cohen, RS Bandurski 1978 Synthesis of C-indole-3-acetyl-myg-inositol. J Labelled Comp Radiopharm 15: 325-329 Nurmann, G, D Strack 1979 Sinapine esterase. 1. Characterization of sinapine esterase from cotyledons of Raphanus sativus. Z Naturforsch 34c: 715-720 Park, JH, BP Meriwether, P Clodfelder, LW Cunningham 1961 The hydrolysis of p—nitrophenyl acetate catalyzed by 3-phosphoglyceraldehyde dehydrogenase. J Biol Chem 236: 136-141 Pengelly, WL, PJ Hall, A Schulze, RS Bandurski 1982 Distribution of free and ester indole-3-acetic acid in the cortex and stele of the Zea mays mesocotyl. Plant Physiol 69: 1304-1307 Redemann, CT, SH Wittwer, HM Sell 1951 The fruit-setting factor from the ethanol extracts of immature corn kernels. Arch Biochem Biophys 32: 80-84 Riov, J, MJ Jaffe 1973 Cholinesterases from plant tissues. 1. Purification and characterization of a cholinesterase from mung bean roots. Plant Physiol 51: 520-528 Scandalios, JG 1968 Genetic control of multiple molecular forms of catalase in maize. Ann N Y Acad Sci 151: 274-293 Scandalios, JG 1969 Genetic control of multiple molecular forms of enzymes in plants: a review. Biochemical Genetics 3: 37—79 Scandalios, JG 1974 Isozymes in development and differentiation. Annu Rev Plant Physiol 25: 225-258 Schmidt-Stohn, G, P Wehling 1983 Genetic control of esterase isoenzymes in rye (Secale cereale L.). Theor Appl Genet 64: 109-115 Schwartz, 0 1960 Genetic studies on mutant enzymes in maize: synthesis of hybrid enzymes by heterozygotes. Proc Natl Acad Sci 46: 1210-1215 Seeley, RC, CH Fawcett, RL Wain, F Wightman 1956 Chromatographic investigations on the metabolism of certain indole derivatives in plant tissues. Ig_RL Wain, F Wightman, eds, The Chemistry and Mode of Action of Plant Growth Substances. Butterworths Scientific Publications, London, pp 234-247 57. 58. 59. 60. 61. 62. 63. 21 Sell, HM, SH Wittwer, T Rebstock, CT Redemann 1953 Comparative stimulation of parthenocarpy in the tomato by various indole compounds. Plant Physiol 28: 481-487 Strack, D, G Nurmann, G Sachs 1980 Sinapine Esterase. II. Specificity and change of sinapine esterase activity during germination of Raphinus sativus. Z Naturforsch 35c: 963-966 Thimann, KV 1935 On the plant growth hormone prodUced by Rhizopus suinus. J Biol Chem 109: 279-291 Ueda, M, RS Bandurski 1969 A quantitative estimation of alkali-labile indole-3-acetic acid compounds in dormant and germinating maize kernels. Plant Physiol 44: 1175-1181 Ueda, M, RS Bandurski 1974 Structure of indole-3-acetic acid myoinositol esters and pentamethylmyoinositols. Phytochem 13: 243-253 Went, FW, KV Thimann 1937 Phytohormones. The Macmillan Co., New York Zimmermann, PW, AE Hitchcock, F Wilcoxin 1936 Several esters as plant hormones. Contr. Boyce Thompson Inst 8: 105-112 EXPERIMENTAL I PARTIAL PURIFICATION OF IAA-MlQ-INOSITOL HYDROLASE FROM.Z§A MAYS L. VEGETATIVE TISSUE 22 ABSTRACT An enzyme which hydrolyzes IAA-myg-inositol (IAInos) was observed in extracts of shoots of dark grown seedlings of ZEEHEEXE L. This enzyme activity, which we call IAInos hydrolase, was partially purified by chromatography over w-aminohexyl agarose, hydroxylapatite and Sephadex G-100, and has an apparent molecular weight of 45,000 D. The enzyme preparation can also hydrolyze a-naphthyl acetate. A second esterase was also partially purified from extracts of maize seedlings. This esterase could not catalyze the hydrolysis of IAInos but did hydrolyze a-naphthyl acetate. The hydrolysis of both IAInos and a-naphthyl acetate can be detected in extracts of roots and of germinating kernels as well as shoots. 23 INTRODUCTION Esters of the plant growth hormone indole acetic acid (IAA) serve as one source of the hormone in both endosperm (10,31) and vegetative tissues of Zga_mgy§ (7). High concentrations of esterified IAA relative to that of the free acid have been observed in both vegetative and endosperm tissue (1,2). According to the hormonal homeostasis theory (8), hydrolysis of esterified IAA in any tissue releases free IAA which is then available to the plant as required for growth and response to environmental stimuli. This hypothesis predicts that an enzyme (or enzymes) will be present in vegetative tissue which catalyzes the hydrolysis of esters of IAA. This paper reports the partial purification of an enzyme which catalyzes the hydrolysis of IAInos. A preliminary report has appeared (12). IAInos was chosen as substrate for lg yitrg analysis of IAA ester hydrolase activity for the following reasons. First, IAInos is present not only in the endosperm of 2. E215 kernels (19) but is also found in shoots of 4 d dark grown ;. may§_seedlings (6). Second, IAInos constitutes almost 20% of the ester pool of both endosperm (10) and vegetative tissue (6). Third, experiments measuring the transport of radioactive IAInos indicate that IAInos is transported from seed to shoot in quantities sufficient to support normal elongation growth in a young corn shoot (22). Finally, IAInos was available as a radioactive compound (20) and by chemical synthesis (23). 24 25 Plants contain many esterolytic enzymes (4,11,13,15,17,18,24,26, 29,30), but not much is known about esterase specificity. For example, it was not known whether any of the several esterases reported in 2, EEZE (28), would be able to hydrolyze IAInos. Very often esterases have been characterized on the basis of their ability to hydrolyze synthetic substrates (4,14,15,27). In our work the hydrolysis of a synthetic ester, a-naphthyl acetate, was studied to see if an enzyme fraction hydrolyzing IAInos was also active in the hydrolysis of a general esterase substrate, and, conversely, to identify other esterases which could then be examined for activity in the hydrolysis of IAInos. If all esterases were capable of catalyzing the hydrolysis of IAInos then the problem of how such activities are regulated would indeed be complex. If only one or a few enzymes catalyze IAInos hydrolysis, then it is more feasible to look at the mechanisms underlying regulation of the activity. Our purification of esterase activities demonstrated that not every esterase has IAInos hydrolyzing activity. We report that at least one esterase can be partially purified from shoots of ;. may; seedlings, and that it cannot hydrolyze IAInos. We also report that IAInos hydrolase activity is not unique to shoot vegetative tissue, but is also present in seed and root tissue. This work is one of few reports in the plant biology literature (11,17,18,24,26,29,30) in which an esterase hydrolyzing an endogenous ester substrate has been purified and characterized and is to our knowledge, the first report of the partial purification of an enzyme catalyzing hydrolysis of an IAA conjugate. MATERIALS AND METHODS Chemicals The following chemicals were obtained from commercial sources and were used without further purification: Fast Violet B, Coomassie Brilliant Blue R and w-aminohexyl agarose were from Sigma; Bio-Gel HTP and bovine plasma gamma globulin from BioRad; Sephadex G-100 superfine from Pharmacia; ACS Scintillation cocktail from Amersham; and (NH4)2504, enzyme grade from Mann Research Laboratories, Inc. [5-3HJ-indolyl-3-acetyl-myg-inositol (29 Ci/mmol) was synthesized as reported by Michalczuk and Chisnell (20) and was a gift from J. Chisnell. a-Naphthyl acetate was synthesized from a-naphthol and acetyl chloride in benzene (3) and recrystallized from 2-propanol. Preparation of Plant Material Kernels of 1. may; sweet corn (Stowell's Evergreen, Burpee Seed Co.) were imbibed in running tap water overnight, then depending on the amounts of enzyme required, either sown in vermiculite in flats or rolled in paper towels, and grown for 4 d in darkness at 25°C. Shoots, including mesocotyl, coleoptile, and primary leaves, were harvested under a green safe light and collected in an ice chilled beaker. After weighing, an acetone powder was prepared from the shoots by grinding in a blendor in acetone (10 ml of acetone to 1 9 tissue) chilled to -5 to -10°C with dry ice. The acetone was removed by filtration and the 26 27 residual powder rinsed with cold diethyl ether. The resulting powder was dried over P205‘1n‘ygggg and stored at -18°C. Root extracts were obtained by grinding primary roots in cold Tris. Liquid endosperm was collected in chilled Tris by nicking, then squeezing kernels of 4 d seedlings. An acetone powder made from whole kernels of 4 d seedlings was kindly provided by H. Nonhebel. Buffers Tris chloride (Tris) and potassium phosphate (Kphos), both at 0.05 M and pH 7.15 were used except as noted. Protein Concentration Protein was determined by the method of Bradford (3a) using bovine plasma gamma globulin as the standard. Polyacrylamide Gel Electrophoresis Electrophoresis was conducted on slab gels 2 mm thick using a discontinuous buffer system (25). The stacking gel (4.5% acrylamide) was in 0.75 M Tris, pH 7.0, and the separating gel (8% acrylamide) was in 3 M Tris, pH 8.9. The reservoir buffer was Tris-glycine (2.4 mM Tris, 24 mM glycine), pH 8.3 (9). Proteins were stained with 0.1% (w/v) Coomassie Brilliant Blue R in acetic acid:methanol:water (5:20:75) and destained in the same solvent. Esterase Activity Staining Following electrophoresis gels were rinsed with Tris. a-Naphthyl acetate (40 mg in 4 ml of 50% aqueous acetone) was added to a chilled 28 mixture of 200 ml of Tris containing 100 mg of Fast Violet B. After mixing the solution was poured over the drained gel and incubated at room temperature until bands of the desired intensity developed (approximately 10 to 20 min). In some cases the gel was soaked 20 to 30 min in 0.5 M boric acid prior to rinsing in Tris since this reduced background discoloration of the gel. After staining, the gels were rinsed with fresh Tris and photographed. Gels could be stored in the cold for 3 to 4 days, or for more permanent storage, gels were dried onto Whatman 3MM filter paper. Assay of IAInos Hydrolase Activity Reaction mixtures contained plant extract plus Tris or Kphos in a total volume of 100 pl in a 6 x 50 mm culture tube (Kimble). Five pl of 3H-IAInos in 50% aqueous 2-propanol (6 pmol IAInos/5 pl, approximately 3 x 105 dpm) was added and a 5 pl aliquot withdrawn immediately for counting. The mixture was incubated at 37°C for 4 h and the reaction terminated by the addition of 100 pl of 2-propanol. A control reaction mixture without plant extract was used to determine non-enzymatic hydrolysis. Reaction mixtures were frozen if not analyzed immediately. In early experiments the extent of hydrolysis was determined by chromatographing the reaction mixture over approximately 1.2 ml DEAE-Sephadex (acetate form in 50% aqueous 2-propanol) in a disposable Pasteur pipette. Unhydrolyzed 3H-IAInos was not bound to the DEAE and was eluted with 4.0 ml of 50% aqueous 2-propanol. The product, 3H-IAA, was then eluted with 5.0 ml of acidified (2N acetic acid) 50% aqueous 2-propanol. Aliquots of 0.5 ml from both the neutral and acidic fractions were used for determination of radioactivity in a Beckman LS 29 7000 liquid scintillation counter using ACS solution as the scintillant. In later experiments the reaction mixture was mixed with approximately 0.5 ml of DEAE-Sephadex in a 1.5 ml polypropylene tube (Cole-Parmer) and the unreacted substrate removed by a series of centrifugations in a Beckman Microfuge B. After each centrifugation the supernatant solution was removed for determination of radioactivity; the exchanger was mixed with 0.3 ml of 50% aqueous 2-propanol and the cycle repeated 6 times. The product, 3H-IAA, was then removed by 5 cycles of centrifugation in a total volume of 1.5 ml acidified 50% aqueous 2-propanol. Recovery of radioactivity averaged 85 to 90% using either method. Assay of a-Naphthyl Acetate Esterase Activity The production of a-naphthol was followed by measuring the increase in absorbance at 320 nm. Reaction mixtures included buffered plant extracts and 4.3 pmol a-naphthyl acetate in a total volume of 3.0 ml. The mixture, excluding substrate, was incubated at 37°C in the specimen chamber of a Cary 15 Dual Beam Recording Spectrophotometer for 5 min. After addition of substrate, the reaction was incubated and observed for at least 8 min. The rate of hydrolysis was determined by measuring the increase in absorbance for the first 5 min of the reaction. The molar extinction coefficient of a-naphthol in this reaction mixture was determined to be 2496 L/mol/cm. This agrees with the value reported earlier by Johnson and Ashford (16). Rates of hydrolysis are reported as nmol a-naphthol produced/min/mg protein. RESULTS Purification The following steps were used in the purification of IAInos hydrolase and were conducted at 4°C. §£§2_l- Preparation of crude extract. Ten 9 of acetone powder was suspended in 150 ml of Tris with gentle stirring for 30 min. Particulate inatter was removed by centrifugation at 12000 g for 10 min, the resulting pellet resuspended in the same volume of fresh buffer and extracted an additional 30 min. Following centrifugation, the supernatant fluids were combined and designated Stage I enzyme. Step 2. Ammonium sulfate precipitation. Solid ammonium sulfate was added to Stage I enzyme to 90% saturation, the mixture stirred 45 to 60 mini, then centrifuged at 12000 g for 15 min. The pelleted protein was crissolved in 60 ml Kphos. The protein solution was dialyzed against 2 (flianges of the same buffer for a total of 3 to 4 h. The resulting extract, Stage II enzyme, could be frozen and would retain hydrolase ac1:ivity for as long as 1 year. Upon thawing a precipitate formed which cOLFld be removed by brief centrifugation with no loss of activity. Step 3. Hydrophobic interaction chromatography. Clarified Stage II enzyme was chromatographed over an ur-aminohexyl agarose column eqtrilibrated with Kphos. The column was washed with the same buffer unti'l protein was no longer detected by absorbance at 280 nm in the effluent. Protein bound to the column was eluted with the starting 30 31 buffer containing 0.5 M NaCl. The column was regenerated by washing with the starting buffer. Most of the IAInos hydrolase activity was not retained on the column, and appeared in the first protein peak (Figure 1). Approximately 70% of the IAInos hydrolase activity applied was recovered between 11 and 40 ml. These fractions were pooled and ammonium sulfate added to 90% saturation. Precipitated protein was resuspended in 35 ml 0.01 M Kphos, pH 7.15, dialyzed against 2 changes of the same buffer and frozen. Hydrolase in the frozen suspension remained active at least 2 months. After thawing, a precipitate was observed which was removed by centrifugation with no loss of activity. This fraction was designated Stage III enzyme. Step 4. Hydroxylapatite chromatography. Thirty ml of clarified Stage III enzyme was chromatographed over hydroxylapatite prepared in 0.01 M Kphos. Unbound protein was eluted with the same buffer until the absorbance of the eluate at 280 nm decreased to less than 0.1; protein bound to the column was eluted with a gradient of Kphos from 0.01 M to 0.5 M, pH 7.15. IAInos hydrolyzing activity eluted after the gradient was applied. Tubes containing hydrolase activity were pooled and stored at -18°C. This fraction was designated Stage IV enzyme. When the column was overloaded, some enzyme activity appeared tailing the peak of unbound protein. The purification by hydroxylapatite chromatography was only 1 to 3 fold, and the recovery of total activity was about 50%. The unbound protein fractions were active in the hydrolysis of a-naphthyl acetate. These fractions were pooled as above, and were designated as Stage IVE enzyme. 32 (Pu-noun!) VVl-Hg .— - 0.20 d 0.l0 d 0.05 40.5 (paw-Town) ‘lOl-lLHdVN-l' o-o (D I) <3 9 s o 8 1 I I T 20 FRACTION NUMBER 5.0 Figure 1. HydrOphobic interaction chromatography of Stage II enzyme. The elution pattern of enzymatic activities of Stage II enzyme from w-aminohexyl agarose column (column size, 1.8 x 5.2 cm; flow rate 0.75 ml/min; fraction size, 10 ml/fraction) is shown. At fraction 10 protein bound to the column was eluted with 0.5M NaCl in Kphos buffer. Fractions were assayed for hydrolase (o) and esterase (0) activity as indicated, using 0.1 ml extract/assay. Fractions 2 to 4 were pooled for further purification. 33 Step 5. Gel filtration chromatography. Stage IV and Stage IVE enzyme preparations could be further fractionated by chromatography over Sephadex G-100. Chromatography of approximately 12 g of protein of Stage IV enzyme over Sephadex G-100 (column size 1.6 x 33 cm, flow rate 10 ml/h, 1 ml/fraction) resolved the mixture as shown in Figure 2. Fractions between 26 and 32 ml were pooled and stored at -18°C for up to one month with no loss of activity. This fraction was designated IAInos hydrolase. Stage IVE enzyme was concentrated by precipitation with (NH4)2S04 and 15 mg was chromatographed over Sephadex G-100 (column size 1.5 x 23 cm, flow rate 10 ml/h, 1 ml/fraction). Figure 3 shows the protein and esterase activity elution profiles. Fractions 10 to 25 were pooled and stored at -18°C. This preparation was designated as estersae. A portion of this esterase was rechromatographed over a calibrated Sephadex G-100 column and was used in some experiments. It is designated esterase R. A flow chart for the purification of IAInos hydrolase and esterase activities is shown in Figure 4. Table 1 summarizes the purification of IAInos hydrolase and Table 2 summarizes the esterase purification. Polyacrylamide Gel Electrophoresis Duplicate samples from different stages of purification were separated electrOphoretically. The gel was divided in two and one portion stained with Coomassie Blue, the other portion with a-naphthyl acetate in the presence of Fast Violet B. In this way the mobilities of bands of protein could be compared with mobilities of bands of esterase activity. Prior to chromatography on hydroxylapatite there are up to 12 protein and 8 esterase bands. Not each of the 8 esterase bands has a 34 (pa-Tom) VIII-He H C) 9 3 3. T. 3. <2 <> c> <3 C) (3 <3 ELUTION VOLUME (ml) 0. 0. In. 0. '0 . cu GU " " (3 093v _— Figure 2. Gel filtration chromatography of Stage IV enzyme. The elution pattern of IAInos hydrolase activity of Stage IV enzyme from Sephadex G-100 (column size 1.6 x 33 cm; flow rate 0 ml/h, 1 ml/fraction) is shown. Fractions were assayed for H-IAInos hydrolysis as indicated. Fractions 26-32 were pooled for further experiments. 35 (It! gag) 0.7 . ‘ /ESTERASE n q 70 A T .5 0.6 - .160 E '3 E 5 (315*; 4!5(> _J O I: P 0.4 " V q 40 E ( a z. (N 03 " ‘ 30 i 0.2 - .. 20 0.l IO (1. () IO 20 30 40 50 ELUTION VOLUME (ml) Figure 3. Gel filtration chromatography of Stage IVE enzyme. The elution pattern of Stage IVE esterase from Sephadex G-100 column (column size 1.5 x 23 cm, flow rate 10 ml/h, 1 ml/fraction) is shown. Fractions were assayed for a-naphthyl acetate hydrolysis as indicated. Fractions 10-25 were pooled for further experiments. 36 Acetone Powder Extraction £292; 90% (NH4)2304 Stage_II urAminohexyl Agarose Stage II Hydroxylapatite l 1 Sta e IVE Sta e IV Sephadex G-100 Sephadex G-100 Esterase IAInos ydrolase Sephadex G-100 Esterase R Figure 4. Procedures for the partial purification of esterase and IAInos hydrolysis. This flow chart shows the procedures used to partially purify the esterase and IAInos hydrolase as described in the text. 37 ma ea me Nm ooH Auv excm>oomm .mechm H macaw eo »u_>_uo< eo wave: pouch + xuw>_po< mo mu_:: Poncho .me»~cm H mmmpm mo auw>Puo< u_wwumam * »u_>_uo< o_$_umamo .:_muocm _muo» x »u_>_uu< o_wwumamn .mocHwuoe we muwcse m.m Am ¢.H mm 8.3 mm H Hofl H mmfl n»o_>wou< to oeo_oao_eweaa mp_e: _aooe .mmm—oeu»; mocHPou< Amev A_ev u_w_uwam :wmuoca peace mas—o> .H u4m

_ HHH HH cowuumcu 38 .mEHNcm H macaw mo AuH>Hpo< mo muwcz Hugo» * xpp>Huo< Ho maps: Hmuopu .mechm H macaw mo »u_>_gu< o_w_umam * xpw>wuu< UHHHumamo .=_maoga HmHOH x HHH>HH0< UHHHUmqmn .mumpmum —>:pzqmc-v Hos: m.¢ Ho mocmmmga mzu :_ omammmm Hcpmuogq me\;\Hosc qu>Huum we mu_::m mmmHoLux: m H.o wom.¢ m¢ mm mm ooH-u xmumsaom mo=HH m o.m oom.m coo m NN ooH-w xmumgamm a mmmgmumm H m.H wom.m wmm HH HH ooH-w xmuwxamm wmmgmumm MN H.m mom.~m Hov Hm mm wuHHmamHonguxr H>H omogmmm OH ¢.H cow.mm mm ch mm Haxmgo=_e<-a HHH mm m.H moo.wNH mm mva Ho HOWNHHIZH mom HH ooH H owm.mmH OH HmmH «mm gonzo; mcoumu< H Hxv DHHH>HHU< Ho QHHH>HH0< Hmev HHEH uxgw>oumm ocOHHmuHHHLza mpwcs chop UHHHUmam chuoLm Hugo» msaHo> acmsymmgh :o_uumgu .mmmgmumm mo :oHumUHHHL3a .m HHmHocuxz mumumu< Hxspsgmzua ooH.o mm~.o omo.o moo.o :Hmuoca me\;\HoEq umeHocux: mocHHHum uHumE»~:m we comHLquoo .m m4m

coo mew: :_mpocq as Ema mmummn .gmewsa m_ch HE o.m :_ uwuumcpxm mm: gonzoa «acumen m H.o Hana Hamoxm HNHV umorcumoc mo umcmgwca mm: mex~cm H mmaumm «a o p we Hoo.o Noo.o H moo.o WHHHouomaz oHH m a He omo.o moo.o A NHo.o mmHHHQooHou Queen uccHa\;\Ho5a :kuoca me\;\HoEa nucmq acmHa\z\HoEa chuoLq ms\;\HoEa AHH>Huu< mumumu< HazugamZIa xaw>Huu< mmmHocuzz mocHHpuw UHHmex~cm Ho :ochwaeoo .N HHmcmmno pumtmm o: o mmeHocu»: mo man; so um>gmmao uummmm + o + o mlonH.¢ u: o u: mion~.H O + + QIOHXHoN HocmoxHo mcoHpmu a:o_;HaH=Hw co muuacem Dex .mmmHocu»: mocH apacpmaam mHm nEx wx mE> ooo.ma oo0.HN aHaEHHmH 3: mmmHocuxx mmmcmamm uumcuxm muocumnsm .mmcHocuxg mocHHuuc Hocucoo Ho Hampton mm HHH>Huu we we ooH ooH HH om amH ooH aex~=a H macaw - HH ooH ooH mHH am NmH ooH awapacamoga scam Haas: Laz+ Luz. +~mz+ Hmz+ +~mz+ Hocucou +~mz+ sz+ +~mz+ Hocucou mxu_>_uu< mmmcmumm mpo>Huu< encumzamoza pocchm .mHmHHocaH; auapaua szpgaac-a Ho JUHLO=HH Ha =o_H_a_;:H .H HHmHHU< aHaHau< ngggaaz-a moo.o mmo.o woo.o mmo.o mHo.o omo.o Hoo.o mNo.o cAHLma u:m_a\z\HOEqv Noo.o A moo.o moo.o H mHo.o Hoo.o H moo.o moo.o H mHo.o Hoo.o H «Ho.o moo.o w NHo.o Hoo.o H woo.o Noo.o H oHo.o HchHoLa me\:\HoEQV HHH>HHU< mmmHoLuxz mocHcw Ho muHsmmL mo meEE:m .H HHmmcu quouommz mHHuaomHou mace—a umuwmcp quouomwz aHHHgoaHou mpcmHa Hocucou mucmEHcmnxm acmHH 111 .chuoca me\meuouomme mo.H Ho couumH :ongm>:ou m mm>Hm meuouomms cot :oHumHsuHmu LmHHEHm < .cmmwan _E\:Hmuocq as H Hm new .Hgmpm: smock m oH\Lmuzoa mcoumum m ¢.o-m.o Hm .usmHmz gmmcw m\mH»HouommE w HH czocx mm; H_ mHauouommE Lou :Hmuocq me\mmHHugowHou v.0 :Hmuomm ms\mumc mHHpaomHoo\muoc u cmuzoa ms\:HmHoca me m¢.o gmuzon as oooH\mmHHunomHou omH chHoca me\mmHHHQomHou ¢.o u gonzoa me\:HmuoLa as me.o u cwwwwmnHflrmnmwmnWMWfi me . - Hz smote a oH\cauzoa m mH.o cmuzoa m\mmH_uaomHou omH 1 p3 smog» m\mmH_unomHou «H .cmesa HE\:_ouoLq we mH Hm new .pcmHmz :mmcH m oH\mezoa «acumen m w.o-H.o AN .HgmHmz cmmcw m\mmH_uqoumHou eH HH czocx mo: mH mmeuaowHoo Lou .xuz mconHHoH any cH meow mm: quouomwe Lo mHHHQomHoU\mumc o» :kuoca me\mumc soc» HHH>Huum mo concm>coom 112 On the basis of experiments described here coleOptiles have higher specific activity for IAInos hydrolysis than mesocotyls where specific activity is defined as the rate/mg protein. The actual activity per part is also higher for IAInos hydrolysis in coleoptiles. The specific activity for a-naphthyl acetate hydrolysis is about the same for mesocotyls and coleoptiles, although on a per plant part basis coleoptiles are more active. The only really significant change in activity seemed to be in mesocotyls exposed to light. The IAInos hydrolase activity appears to have doubled. When exposed to a gravitational stimulus the IAInos hydrolase activity is slightly decreased. The activity in coleoptiles does not change significantly in either case. I would have predicted that in plants exposed to light, in which growth would be inhibited, activity would decrease. In gravistimulated plants activity might be expected to increase or remain the same. In either case since more activity is observed in coleoptiles, and because we usually think of the tip as the source of free IAA, I would have expected activity to change in that part of the plant rather than in mesocotyls. This kind of experiment needs many repetitions under better controlled experimental conditions before any firm conclusions can be drawn. It is interesting to note that esterase activity does not change. It would be interesting to see if rates of hydrolysis of Me-IAA change. APPENDIX F Synthesis of a-Naphthyl Acetate a-Naphthyl acetate was synthesized by the method of Spassow (1). a-Naphthol (0.1 mol) and 1.2 9 Mg turnings were mixed in 35 ml benzene. Acetyl chloride (0.1 mole) was added and the mixture was refluxed for 1 h. After cooling, 150 ml diethyl ether was added and the mixture transferred to a separatory funnel. The solution was washed with 150 ml water, 150 ml of a saturated solution of NaHC03, and 150 ml water. The ether-benzene phase was dried over anhydrous, granular Na2504, then dried in yagug. The brown oily residue was chilled on ice and precipitated as a beige paste. The precipitate was redissolved in 2-propanol and chilled. The resulting crystals, which were soluble in ethanol and acetone, were redissolved in 2-propanol and chilled. After recrystallization the product was filtered and washed with cold 2-propanol, then dried over P205 under vacuum. The resulting needle-like crystals were stored in a brown bottle at room temperature. The melting point was 45°C. Spassow reported a melting point of 46°C. The product, a-naphthyl acetate, at pH 7.15, has absorbance maxima at 264 and 272 nm and does not absorb at 320 nm. a-Naphthol absorbs maximally at 279 nm with a smaller sharp peak at 320 nm and a shoulder at 305 nm. a-Naphthyl acetate was suspended in Tris at pH 7.5, and the pH was raised to 12 by the dropwise addition of 2N NaOH. After 15 min 2N HCl was added until the pH returned to 7.5. The a-naphthol released by alkaline hydrolysis had a UV absorption spectrum identical to that of authentic a-naphthol at pH 7.5. 113 114 Reference 1. Beilsteins Handbuch Er Organischen Chemie 1966 Vol 6, System Number 2928 Springer-Verlag, Berlin 115 va.o oHHH>HHu< UHHHUaam Hmoow HmH.o ¢.mH m.mw mammom mHeom H.w m.om qmmmmm HRH HHH NaOHHuaELHEQU mquuon> cuHu_u< mxcm>oumm Hmpoh .; m.e uwpmazucp mm: mcsuxpe :oHHummc ms» .HE\chuoca me o.- cm=HmH=ou mHaEmm as» Hexmmm mmmwxw chc\sau Hmuop AcoHpomLH UHuHum .quEmmv HcoHuumLH Hmcuamc .ngEmmV chL\H: omumHasomv AcoHuuuem uHu_um .HocucouvHHu< UHHHomam .He\me o.- no; :oHHmcucmucou :Hmuoca mg» mHnmem m_;u :H .chuoLq me\;\Hoan m. g 4 1 HHocpcouH u_e_u< a 1 HaHHEamH uHo_u< a 1 HHHUOHa> Huacpxa H: om\2;\_osa HmH.o 1 Hose 8 x .umm: mm: Humcuxm mo H: cm can .; m.¢ mm: mEHH :oHumnaucH .mczuxHE :oHHUGmL 8:» cu umuum we: mo=H< emu :o_uumcm uHu_um + emu :oHHumcH Hmcusmz n xgm>oummm .Hmz mamm wsu cH cmuoocu mH meoHHomgH UHUHum cH HHH>HuumoHumm .mcHucaou Ho HucmHuHHHm LOH umuumccou new .mHaEmm some L04 uchuou mH mcowuumcm Hugusm: :_ HHH>HpoooHumm~ .mczuxHE :oHuomwc H: ooH\sau w>Hm on vmuumggoo use mcHuczoo Co mocmHQHHHw to» uwuumccou .umgzmmme mp mczuxHE cowuummc zoom EoLH uozcme H: m a c_ »u_>_uumopummH APPENDIX H Calculations Used to Determine the Amount of a-Naphthol Produced by Hydrolysis of a-Naphthyl Acetate The change in absorbance at 320 nm (AA320) is measured between 100 and 400 seconds. (I) AA320/min = AA320/3OO sec x 60 sec/min (2) Concentration (C) nmoles a-naphthol produced/min/volume of extract A /min .—§§%§3—— x 3.0 ml x 10-3l/ml x 109 nmol/m0] from Beer's Law Concentration (C) =-§% where b = cm and E = 2496 I/mol/cm for a-naphthol at pH 7.5. Sample Calculation for a reaction which contained 50 pl Stage III enzyme. The AA320 = 0.195/300 SEC. (1) AA320/min = 0.195/300 sec/50 p1 x 60 sec/min = 0.039/min/50 pl (2) C = 2492.?735T7245311cm x 3.0 ml x 10'3l/ml x 109 nmol/ml 46.9 nmol/min/50 pl 117 LIST OF REFERENCES LIST OF REFERENCES Axelrod, B 1948 A new mode of enzymic phosphate transfer. J Biol Chem 172: 1-13 Avery, GS, Jr, J Berger, B Shalucha 1941 The total extraction of free auxin and auxin precursor from plant tissue. Amer J Bot 28: 596-607 Avery, GS, Jr, PR Burkholder, HB Creighton 1937 Avena coleoptile curvature in relation to different concentrations of certain synthetic substances. 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