1m WWW 1/ , WW 3 1293 00836 0186 }V4531_J BEIURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from .‘nnuzsnuun. your record. FINES will be charged if book is returned after the date stamped be10w. quY 0 519933 1 ‘ ______-._—-—--—— THE CATABOLISM OF INDOLE-3-ACETIC ACID TO OXINDOLE-3-ACETIC ACID BY ZEA MAYS BY Dennis M. Reinecke 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 1987 ABSTRACT THE CATABOLISM OF INDOLE-3-ACETIC ACID TO OXINDOLE-3-ACETIC ACID BY ZEA MAYS BY Dennis M. Reinecke Previous studies have shown that the plant hormone indole-3-acetic acid was oxidized to oxindole-3-acetic acid in endosperm tissue of Zea mays (corn). This study demon- strates for the first time that oxindole-3-acetic acid occurs in vegetative tissues of Zea mays, and that enzyme preparations from endosperm and vegetative tissues catalyze this oxidation reaction. Oxindole-3—acetic acid is inactive in promoting growth in Zea mays mesocotyl. Thus, catabolism of the plant hormone indole-3-acetic acid is of interest since it is an irreversible output from the hormone pool which may be involved in regulating the hormone level. Oxindole-3-acetic acid was demonstrated to be a natu- rally occurring compound in both shoot and endosperm tissues 1 1 by an isotope at 49 pmol'shoot' and 357 pmol'endosperm- dilution assay. Oxindole-3-acetic acid occurs in levels similar to that of free indole-3-acetic acid in these tissues. Enzyme extracts of all corn tissues examined including shoot, root, endosperm, and scutellum oxidized indole—3-acetic acid to oxindole-3-acetic acid at rates of 1 1'mg protein-1. The enzyme catalyzing this to 10 pmol'h' reaction has been shown to be heat labile, a soluble enzyme by centrifugation studies, and oxygen requiring by Thunberg tube assays. A heat-stable cofactor or co-substrate, extractable with non-ionic detergent, stimulated the enzymatic reaction up to 10 fold. Cofactors of peroxidase, mixed function oxygenase, and intermolecular dioxygenase did not replace the lipid factor in the enhancement of enzymatic activity. The chromatographic properties of the lipid-soluble factor were similar to that of an unsaturated fatty acid, and the unsaturated fatty acids linoleic and linolenic acids substi— tuted for the lipid factor in the stimulation of oxidation of indole-3-acetic acid. In a model system, soybean lipoxygenase plus linoleic acid oxidized indole-3-acetic acid to oxindole-B-acetic acid. However, corn lipoxygenase activity did not co-frac- tionate with indole-3-acetic acid oxidase activity following ammonium sulfate fractionation, and suggested that the IAA oxidase was a novel enzyme or a multi-enzymatic system. The identification of the enzyme's mechanism of oxi- dizing indole-3-acetic acid, and the fractionation of the enzyme will contribute to our understanding of hormonal homeostasis and possibly how indole-3-acetic acid promotes growth. ACKNOWLEDGMENTS I would like to acknowledge Dr. Bob Bandurski for giving me the opportunity and means to work on this research project. I will always remember the friendly atmosphere in the lab due to Aga, Bob and the brigade of foreign post docs. I would like to thank my family: Carolyn, Joe, Christine, Brian, and my parents Joe and Cora for believing in me during this trial. And lastly, I especially want to thank my best friend and wife Jocelyn Ozga who made my trip to Michigan a great learning experience. Peace. iv TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . v'i'i'i LIST OF FIGURES ° ' ° ' ° ' ° ° ° ° ° ' X LIST OF ABBREVIATIONS ° ° ' ° ° ° ° ' ° ° xiii INTRODUCTION ' ° ' ° ° ‘ ° ° ° ° ' ' ° 1 LITERATURE REVIEW ° ° ' ‘ ° ' ° ' ° '1 A. INTRODUCTION ° ° ' ° ° ° ° ° ° ' 1 B. IAA CATABOLISM ° ° ° ° ° ° ' ' ° '5 1.DECARBOXYLATION PATHWAY ° ° ° ° ° 6 a. IN VITRO STUDIES ° ' ° ° ° '7 2. OXIAA AND DIOXIAA PATHWAY ° ° ' ° 12 a. IN VIVO STUDIES ° ° ° ° ° ° 15 b. IN VITRO STUDIES ' ' ° ° ' '28 C. PHYSIOLOGICAL OCCURRENCE OF PATHWAYS ° ° 29 D. SUMMARY OF IAA CATABOLIC PATHWAYS ' ° ' 32 E. REACTION MECHANISM FOR THE OXIDATION OF IAA TO OXIAA ' ' ' ° ° ° ° ° ° ' 33 LITERATURE CITED ° ° ° ° ° ° ° ° ° AO EXPERIMENTAL ' ' ' ° ° ° ° ' ‘ ° ° ' ' no SECTION I . . . . . . . . . . . . “6 OXINDOLE-3-ACETIC ACID, AN INDOLE-3—ACETIC ACID CATABOLITE IN ZEA MAYS ABSTRACT O O O O O O O O O O O ”7 INTRODUCTION ' ° ° ° ' ' ° ° ' 48 MATERIALS AND METHOD ' ' ° ' ° ° ' 50 Plant tissue ' ' ° ° ° ° ’ ' 50 Synthesis of labeled OxIAA ' ° ' ' 5O Extraction of tissue ' ' ' ' ' ° 51 CC for the determination of Specific Radioactivity ' ' ° ° ° ' ° 52 Detector sensitivity ' ' ' ° ° ° 56 RESULTS 0 O O O O O C O O O O 57 DISCUSSION ° ' ’ ° ° ° ° ‘ ° ' 61 LITERATURE CITED ' ° ' ° ' ° ’ ' 63 SECTION II C O O O O O O O O O O O 65 OXIDATION OF INDOLE-3-ACETIC ACID TO OXINDOLE-3- ACETIC ACID BY AN ENZYME PREPARATION FROM ZEA MAYS ABSTRACT O O O O O O O O O O O 66 INTRODUCTION ° ° ° ' ° ° ° ° ° '67 MATERIALS AND METHODS ° ° ° ° ° ' '69 Plant materials ° ' ° ' ° ' ' '69 Enzyme preparation ' ' ' ° ' ' ’69 Enzyme assay ‘ ' ° ° ' ' ° ' '70 Validation of assay ' ' ' ' ° ' 72 RESULTS . . . . . . . . . . . 75 DISCUSSION ‘ ' ' ° ' ° ° ' ' ° 90 LITERATURE CITED ° ' ° ° ' ' ' ' 93 SECTION III ’ ' ° ' ' ° ' ' ' ° ' 9S ENHANCEMENT OF INDOLE-B-ACETIC ACID OXIDATION TO OXINDOLE-3-ACETIC ACID BY A LIPID COFACTOR FROM ZEA MAYS ABSTRACT . . . . . . . . . . . 96 INTRODUCTION ° ° ' ' ' ° ° ' ° '97 MATERIALS AND METHODS ° ° ° ' ° ° '99 Plant materials ’ ° ° ° ° ' ' '99 vi Enzyme preparations ° ' ° ° ' ' 99 Extraction of lipid factor ' ° ' '100 Enzyme assays ' ' ' ' ° ' ° ° 101 Fatty acids ' ' ° ° ° ' ° ° ' 102 RESULTS . . . . . . . . . . . 103 DISCUSSION ' ’ ' ° ' ' ° ° ° ' 11A LITERATURE CITED ° ° ° ° ' ° ° ' 118 APPENDICES APPENDIX A . . . . . . . . . . . . . .120 Enzymatic activity as a function of germination time APPENDIX B . . . . . . . . . . . . . ’126 Tritium exchange experiment APPENDIX C . . . . . . . . . . . . . .135 IAA and OxIAA effect on enzyme activity APPENDIX D . . . . . . . . . . . . . '138 Enzymatic activities of seedling tissues LITERATURE CITED ° ' ' ° ° ' ° ° ° ° ° ’14“ BIBLIOGRAPHY ° ' ° ' ° ' ' ° ° ° ' ° ° 1A6 vii LIST OF TABLES Table Page EXPERIMENTAL I Table 1. A quantitative determination of the amount of OxIAA in Z. mays shoot and endosperm tissues by an isotope dilution method. ' ' ° ' ‘ ' ' 58 Table 2. Amount of IAA and OxIAA in Zea seedlings. ’ '59 EXPERIMENTAL II Table 1. Authentic OxIAA and enzymatically synthesized putative OxIAA have the same chromatographic properties as evidenced by HPLC and GLC. ° ° 78 Table 2. The sedimentation characteristics of shoot and endosperm enzyme preparations. ' ° ° ' ' 81 Table 3. Effect of inorganic ions, and sulfhydryl reagents on the enzymatic oxidation of IAA to OxIAA with endosperm enzyme preparations and Triton X100 endosperm enzyme preparations. ' ' ' ’ '83 Table A. Effect of cofactors and co-substrates of oxygenases and peroxidase reactions on the oxidation of IAA to OxIAA by endosperm enzyme preparations. ' ' ' ° ° 84 Table 5. The biological activity of IAA and OxIAA (10'7 to 3 M) in the enhancement of dark- -grown corn mesocotyls. The initial lengths of the mesocotyl sections were 4.6:0.1 mm, cut 2 mm below the coleoptile node. Eleven to 13 sections were assayed in each treatment, and data is expressed as treatment length minus initial length. ' '89 EXPERIMENTAL III Table 1. Mercaptoethanol, cyanide, and BHT inhibition of viii corn enzyme-catalyzed oxidation of IAA, and the soybean lipoxygenase-catalyzed oxidation of IAA. O O O I O O O O O O O O 0 111 APPENDICES Table 1. The 3H radioactivity associated with IAA and OxIAA at various stages of purification following incubgtion of corn endosperm enzyme preparation with H O and IAA. The IAA recovery for the enzyme assay and the boiled control was within 1% through step 3. The OxIAA recovery for the enzyme assay and the boiled control was within 3% through step 4. . . . . . . . . . . . . . 13“ Table 2. The effect of increasing IAA concentration on the oxidation of IAA. Catabolites synthesized are expressed as nmol/L of assay medium. ' ' ' 136 Table 3. The effect of increasing OxIAA concentration on IAA oxidation. The enzyme preparation did not include Triton X100. ' ' ' ' ' ' ° ' 137 Table 4. Enzymatic activities for coleoptile, apical and basal mesocotyl, and endgsperm tissues. Recovery refers to recovery18f [ C] following incubation of enzyme with [1- CJIAA. ' ' ' ' ’ ' '141 Table 5. Enzymatic activities determined for (a) cortex and stele of corn mesocotyl, and (b) corn endosperm and scutellar tissues. nd = not determined. '143 ix Figure LIST OF FIGURES Page INTRODUCTION Figure 1. Figure 2. Figure 3. Figure A. Figure 5. Figure 6. The "inputs to" and the "outputs from" the IAA pool determine the steady state level of IAA in a particular plant tissue (eg. corn). IAA inputs include: a) de novo aromatic biosynthesis; b) conjugate hydrolysis; and c) transport. IAA outputs include: d) conjugate synthesis; 6) transport; f) oxidative catabolism; and g) IAA "use" during the growth process. ° ’ ' A The oxidative decarboxylation pathway. The peroxidase catalyzed oxidation and decarbox- ylation of IAA has been demonstrated to occur in many plant species. The natural occurrence of some of these catabolites has been shown. ' 9 Non-decarboxylation oxidative pathway. The carboxyl retaining pathway has been shown to occur in Zea mays (1), and Vicia faba (2). In Vicia faba, IAA-asp is oxidized to DiOxIAA-asp and glucosylated to glc-DiOxIAA-asp. Oryza sativa bran contains OxIAA and DiOxIAA and their S-hydroxy analogs, but the precursor- product relationship with IAA has yet to be demonstrated. ' ' ‘ ° ' ' ’ ' ‘ ' '1“ The 70 eV mass spectra of the pentafluorobenzyl ester of authentic oxindole-3-acetic acid (a), and that of oxindole-3-acetic acid isolated from Zea mays endosperm (b). This work gave the first precursor-product and MS data for the non- decarboxylation pathway for IAA catabolism in plants. 0 O O O O O I O O O O .20 The ring numbering system for IAA and OxIAA. 3A Oxidation reactions which may be model systems for IAA oxidation to OxIAA. In reactions 1 to A X the molecules being oxidized may be cyclic compounds, while in reactions 5 and 6 the initial molecule being oxidized is a fatty acid with a cis,cis,1,u-pentadiene structure. ' ’ 38 EXPERIMENTAL I Figure 1. Gas chromatographic elution profile of purified plant IAA. As can be seen, the sample has no contaminants which would interfere with measuring the PFB-IAA internal standard. Under the GC conditions described in the text the retention time of PFB-IAA was two minutes shorter than the retention time of PFB-OxIAA. ' ' ' ' ° '55 EXPERIMENTAL II Figure 1. Figure 2. Figure 3. Linearity of enzymatic activity as a function of incubation time. Enzyme was prepared by tissue homogenization with 0.05 M phosphate buffer plus Triton X100 as described in Materials and MethOdS. O I O O O I O O O O 76 Enzymatic activity following evacuation and gas flushing of Thunberg tubes. Ultrapure argon (99.995. Matheson) was used in these experiments. Preliminary experiments with 99.9% nitrogen showed inhibition of activity up to A fold. '80 Stimulation of enzymatic activity by Triton X100 preparation of enzyme, and by the addition of the Triton X100 heat- stable factor to enzyme prepared without detergent. ' ' ° ' ° 87 EXPERIMENTAL III Figure 1. Figure 2. Figure 3. C18 HPLC of the corn lipid factor, linolenic acid, and stearic acid. The samples were dissolved in ethanol: hexane 1: 1 (v/v) and eluted from HPLC with ethanol. H20: .acetic acid 80: 19 75: 0. 25 (v/v/v). ' ' 105 The effect of increasing concentrations of linolenic, linoleic, and oleic acids on the enzymatic oxidation of IAA. ' ‘ ’ ' ’ ’107 The effect of increasing concentrations of trilinolenin (1,2,3 tri-[(cis,cis,cis)- 9,12,15 octadecatrienoyl]-rac-glycerol), and xi Figure 4. Figure 5. APPENDICES Figure 1. Figure 2. arachidonic acid on the enzymatic oxidation of IAA to OxIAA. ' ’ ° ° ' ' 109 Ammonium sulfate fractionation (0 to 42%, 42 to 53%, 53% to 80%) of lipoxygenase and IAA oxida- tion activities from 4-day-old endosperm enzyme preparations. The totals for IAA oxidation1and lipoxygenase activities were: 1,247 pmol'h' , and 773 AU'min , respectively. ' ° ' ° ' '112 Ammonium sulfate fractionation (0 to 30%, 30 to 42%, 42 to 60%, and 60 to 80%) of lipoxygenase, hydroperoxide isomerase, and IAA oxidation activity. The totals for enzymatic activities in the ammonium sulfate fractions for IAA oxidation to OxIAA, hydroperoxide is merase, and lipoxy- genase_were: 9,619 pmol'h' , 108 AU'min' , 769 AU'min . . . . . . . . . . . . .113 Total enzymatic activity for IAA oxidation to OxIAA and to an unknown IAA catabolite as a function of days of germination. IAA oxidation products were assayed as previously described following a 13 h incubation period. ' ' ' 122 Specific activity of Triton X100 extracted enzyme as a function of days of germination. ° ' 124 xii BHT DEAE DiOxIAA DiOxIAA-asp FID F Wt GC-MS glc-DiOxIAA-asp HPLC IAA IAA-asp MS m/z NAA OxIAA PFB PVP 2,4-D 3H 4-Cl-IAA 7-OH-0xIAA 7-OH-OxIAA-glc LIST OF ABBREVIATIONS butylated hydroxytoluene diethylaminoethyl dioxindole-3-acetic acid (3-hydroxy-2-indolone-3-acetic acid) dioxindole-3-acetylaspartic acid flame ionization detector fresh weight gas chromatography-mass spectrometry 3-(O-B-glucosyl)-dioxindole-3- acetylaspartic acid high pressure liquid chromatography indole-3-acetic acid indole-3-acetylaspartic acid mass spectrometry mass to charge ratio naphthaleneacetic acid oxindole-3-acetic acid (2-indolone-3-acetic acid) pentafluorobenzyl polyvinylpyrrolidone 2,4-dichlorophenoxyacetic acid tritium 4-chloro-indole-3-acetic acid 7-hydroxy-oxindole-3-acetic acid 7-hydroxy-oxindole-3-acetic acid -7'-O-B-D-glucopyranoside xiii 14 16 18 carbon 14 carbon 16 oxygen 18 xiv INTRODUCTION LITERATURE REVIEW A. INTRODUCTION Indole-3-acetic acid (IAA) is a ubiquitous plant hormone (plant growth regulator) belonging to the class Of plant hormones called auxins. The naturally occurring auxins include indole-3-acetic acid, phenylacetic acid (68), 4-chloro-indole-3-acetic acid (present in Leguminosae, tribe Vicieae (41) (10)), and their amino acid and sugar con- jugates (4). Indole-3-acetic acid was the first plant hormone to be chemically characterized (25), and its history can be traced to the 1930's work on the "growth promoting substance" from £1232 coleoptiles (67). Exogenous IAA has many effects on plant growth including cellular effects on: 1) cell elongation, 2) cell division, 3) cell differentiation; and morphological effects in: 1) geotropism, 2) apical dominance, 3) leaf abscission, 4) and fruit set (of 3). The mode of action of IAA includes short and long term effects including wall acidification and loosening, and nucleic acid metabolism (42) (57). How auxins affect the cascade of events which lead to plant growth and differentiation is not resolved. Understanding auxins is also complicated by the fact that plant hormones 2 can modify the amount and action of other hormones, eg. IAA stimulates the synthesis of ethylene, a hormone which can antagonize IAA action. The isolation and characterization of IAA mutants (59) will be an important tool in unravelling the mystery of a simple molecule having diverse effects on plant growth. Determining the role of endogenous IAA in plant growth has been complicated by the low amount of IAA in vegetative tissues, 15 to 35 ng'g fresh weight-1 (90 to 200 pmol'g fresh weight-1 for corn, peas, and oats (4)), and the lability of IAA during extraction. There is no simple method for the quantitation of endogenous IAA since all methods require a multi-step purification of IAA. Selected ion monitoring gas chromatography-mass spectrometry with a heavy isotope of IAA as an internal standard affords the most reproducible and unequivocal method. The use of an internal standard several mass units heavier than IAA was introduced by Magnus et al. (30) by synthesizing 4,5,6,7 tetradeutero IAA, and recently was improved by the synthesis of ring-labeled 13C IAA (7). One approach to understanding the role of endogenous IAA in growth has been the study of the steady state level of the hormone, and its regulation by the "inputs to" and the "outputs from" the IAA pool (fig. 1) (5). Inputs to the pool include: 1) de novo synthesis, 2) conjugate hydrolysis of ester or amide conjugated IAA, and 3) transport to actively growing tissues. Outputs from the IAA pool Figure 1. The "inputs to" and the "outputs from" the IAA pool determine the steady state level of IAA in a particular plant tissue (eg. corn). IAA inputs include: a) de novo aromatic biosynthesis; b) conjugate hydrolysis; and 0) transport. IAA outputs include: d) conjugate synthesis; 8) transport; f) oxidative catabolism; and g) IAA "use" during the growth process. _ouuo:_<_ nuance—S c.9933 $2333 3a -<<_xo-zo-k F _ £35.33:— utoEoco tonnes. / / 753-10-; / £23-42. I l / G <51 3.933 iau®x3czzé : I o z o z ,¢_\ \ // / _ i _ fut G .3381. O of I No 2 \ Alli _ _ Bum o:>xca..mu-n-o_ouc_ ovasmuEAOEuE .acmme-m-o_ouc_‘\ IOUNIU. n z z / o B 1 8 1 ad :08 0:0 :ofox \ .N manual 10 oxygen in the 3—methyleneoxindole ring originates from water and not molecular oxygen. The additional information that labeled indole-3-methanol is not metabolized by horse radish peroxidase to 3-hydroxymethyloxindole demonstrates that oxindole and indole formation are separate branches of the peroxidase pathway. It is thought that peroxidase initially reacts with IAA by forming an IAA free radical which is subsequently attacked by oxygen (18). Following decarbox- ylation of the IAA, the reaction proceeds either by the indole-3-aldehyde route or the 3-methyleneoxindole route. As mentioned previously, cofactors for IAA oxidation may include Mn++, and monophenols. Interestingly, Mn+++ will non-enzymatically oxidize IAA, and it is thought that peroxidase plus monophenol oxidizes Mn++ to Mn+++ (6). The Mn+++ may then oxidize IAA with the subsequent decarbox- ylation of IAA. Monophenols and m-diphenols stimulate IAA oxidation, while p-diphenols, o-diphenols, coumarins, and polyphenols inhibit the enzyme reaction. In vivo regula- tory functions for these compounds in peroxidase oxidation of IAA have been suggested (53). Care must be taken in interpreting the results of in 13359 oxidation of IAA. After feeding [1”CJIAA (1.6 x 10'5 M) to crude extracts of Scots pine shoots, no IAA remained after a one hour incubation period. Five catabolites, including indole-3-methanol (the major product), were synthesized. When [1-1uCJIAA was incubated with the shoot extract, only 3% of the radioactivity remained in two minor 11 carboxyl-retaining compounds. However, when [2-14CJIAA was fed to isolated Scots pine protoplasts for 5 hours most of the IAA remained unmetabolized (56). With protoplasts the metabolic profile was very different since only 2 catabo- lites were observed, and the major unidentified product retained its carboxyl group. The minor peak was identified to be indole-3-methanol by GC-MS. The in vitro experiments gave evidence for a rapid IAA catabolism via peroxidase catalyzed decarboxylation, while the more physiological protoplast experiments gave evidence for a slower catabolism by a carboxyl-retaining pathway, and a minor role for the peroxidase pathway. Conjugation of IAA with sugars and amino acids appears to protect IAA from peroxidative attack. Purified horse radish peroxidase, and pea and corn peroxidase preparations oxidized IAA rapidly, but not ester and amide conjugates (including indoleacetyl-myo-inositol, indoleacetyl-L- aspartate, indoleacetyl-glycine, L and D indoleacetyl- alanine, and indoleacetyl-B-alanine) (8). The reaction mixture contained dichlorophenol and manganese. Hinman and Lang (18) reported that the ethyl ester of IAA was suscep- ++ tible to peroxidative attack when H 0 Mn , and dichloro- 2 2’ phenol were included in the enzyme assay, and inactive to oxidation in enzyme assays without H O The stability of 2 2' IAA conjugates to peroxidase in the presence of H202 needs to be evaluated. 12 2. OXIAA AND DIOXIAA PATHWAY The oxidation of IAA to OxIAA and, IAA to DiOxIAA will be discussed in this section as one pathway since both reactions oxidize the indole ring and retain the IAA side chain. This oxidation pathway has been observed in several plant species including rice, corn, and broad bean (fig. 3). Colorimetric assays indicate that OxIAA may also occur in germinating seeds of Brassica rapa, and developing seeds of Ribes rubrum (23). [1-1uCJIAA feeding studies gave evidence that the carboxyl-retaining pathway may also occur in Scots pine protoplasts (56), and crown-gall and pith tobacco cultures (64). The standard methods for monitoring the peroxidative decarboxylation of IAA may not distinguish between the two pathways in an in vitro system. For example, IAA oxidation to oxindole-3-acetic acid would result in 02 uptake, the loss of the Salkowski indole color reaction, and the reduction in the 280 nm indole spectrum with an increase in the 247 nm oxindole spectrum-- characteristics positive for the peroxidase system. Measurement of 1uC02 evolution from feeding [1-1uCJIAA is a clear indication of the peroxidase oxidative decarboxylation pathway. Oxindole-3-acetic acid has been shown not to be an intermediate or a substrate for the peroxidase pathway (18) so the pathways are independent. Careful chromatographic isolation and physicochemical identification of the Figure 3. 13 Non-decarboxylation oxidative pathway. The carboxyl retaining pathway has been shown to occur in Zea mays (1), and Vicia faba (2). In Vicia faba, IAA-asp is oxidized to DiOxIAA-asp and glucosylated to glc-DiOxIAA-asp. Oryza sativa bran (3) contains OxIAA and DiOxIAA, and their 5-hydroxy analogs, but the precursor- product relationship with IAA has yet to be demonstrated. 14 O.U< U.hWU< IMINJOOZCAORTIO muIO-LC O.U< U.hwu<1 Mimi—ODEXOIIO mqum I O 2 Ai 90¢ 0.50.. in 300535-75-.. 93. 250(- m- 3002...? -...7. 7. 7.00:6 ._. 3 CI 35830 90¢ o.»mo<-n-m._ooz_xo-zos o- -mmooa..o :owfomvz o a_0< 0...m0<:m:m...002.x0 i l Ionwfl IUI IOW... O~IUHIH. .m 0.33”. 15 catabolites is the best method to identify the IAA catabolic pathways occurring in a particular plant. a. IN VIVO STUDIES The study which led to the search for a carboxyl- retaining pathway for IAA catabolism in corn was an indole turnover study (11). Epstein et al reported that the free IAA pool (308 pmol) turned over rapidly at 96 pmol'h'1. Since the level of free IAA remains essentially constant in these tissues during this stage of development, both the synthesis and destruction of the hormone equaled 96 pmol'h' 1. At the time of the study it was accepted that IAA was catabolized in plants (including corn (6)) by decarbox- ylation. However, when [1-1uCJIAA was fed to the endosperm 1 of corn, only 12 pmol'h' or 12% of the turnover could be 14 accounted for by CO trapping experiments. The authors 2 interpreted these results as indicating that corn may have an alternative pathway of IAA catabolism with carboxyl retention. Nonhebel confirmed these results, showing that IAA was metabolized rapidly without carboxyl loss by corn root and shoot segments (37) (38). The re-examination of the IAA turnover study's data showed that the turnover of IAA was overestimated. The apparent first order rate constant for IAA turnover was determined by feeding radiolabeled IAA and measuring its decrease in specific activity over time. The first order rate constant was calculated from the equation: 16 C kt log -5)= 777—— Ct 2.303 where Co=initial specific radioactivity, Ct=final specific radioactivity at time t, szirst order rate constant, and t=time. The half life of the IAA was determined from the equation: The half life of IAA in corn endosperm tissue was estimated to be 3.2 h for the 308 pmol of endogenous IAA. Errone- ously, the turnover of IAA was calculated to be 308 pmol IAA/3.2 h half life 2 96 pmol/h. However, the first order rate equation is a log equation, and the amount of compound at any particular time is correctly calculated from the rate equation: CO kt log --- = ----- , k:0.217, t=1 h Ct 2.303 C or -E = 0.805, where 80.5% of the IAA remains after 1 h. C 0 Since the pool size of IAA is 308 pmol'endosperm'1, 60 1 of IAA is catabolized per endosperm (308 1 pmol'h' pmol‘endosperm' X 80.5% 'h'1). The level of IAA remains essentially constant during 4 days of germination, so 60 1 pmol'h' of IAA is also synthesized to maintain the steady state level of free IAA at 308 pmol. The actual turnover of IAA at 60 pmol'b‘1 pmol'h'1. is 38% lower than the paper's reported 96 17 The amount of decarboxylation included in the IAA turn- over was measured by Epstein et al by feeding [1-1uCJIAA to 4-day-old seedlings. Twenty-eight ng of [1-1uCJIAA (20,750 DPM) was applied to each kernel via the cut endosperm. Sixty-nine per cent of the endosperm including 37.2 ng of endogenous free IAA (53.9 ng IAA'endosperm'1 X 69%) remained with the seedling after the [1uCJIAA application. Assuming uniform mixing of the labeled IAA with the endogenous IAA in the liquid endosperm of the seedling, the specific radio- activity of the IAA was: 20,750 DPM --------------- = 318 DPM/ng 28 ng + 37.2 ng In 9 experiments with 8 replicates, the average decarbox- ylation was 1800 DPM'8h'1. The amount of decarboxylation per hour can be estimated to be: 1800 DPM ng nmol 4 pmol 8 h 318 DPM 175 ng h 1 1 (Epstein et al reported that there was 12 pmol’h' 'plant' using the same equations and the same data, with the exception that they incorrectly used a value of 175 ng as the endogenous level of IAA.) The recalculations of the Epstein data show that IAA was turning over at 60 pmol'h'1, and that decarboxylation 1 or 7% of the IAA turnover in endosperm of corn seedlings. Fifty-six pmol°h"1, or 93% of accounted for 4 pmol’h' the IAA turnover was not accounted for by decarboxylation. 18 Thus, the authors' conclusion that another pathway for IAA catabolism might occur in corn was valid. To test this hypothesis, Reinecke (43) incubated corn seedlings with [1-1uCJIAA via the endosperm. By a series of extraction and purification steps, a carboxyl-retaining IAA catabolite was isolated. The catabolite had the same chromatographic properties and mass fragment ions as oxindole-3-acetic acid (fig. 4). This was the first physicochemical report of the precursor-product relationship for the oxidation of IAA to OxIAA in plants. IAA transport studies led to the discovery of conju- gates of dioxindole-3-acetic acid (DiOxIAA) in the roots of Vicia faba (60). Feeding through nutritive tissues (cotyledons of V. faba and endosperm of Z. mays), a more physiological approach to applying hormone than through earlier methods of application through cut shoots or across the epidermis of intact shoots and roots, may explain partly the hiatus of 25 years between the discovery of IAA catabo- lism and the discovery of the carboxyl-retaining pathway. The availability of relatively inexpensive radiolabeled IAA also facilitated the discovery of the new pathway. Isotope dilution experiments (44) showed that OxIAA was a naturally occurring compound in Zea mays endosperm and 1 and 47 pmol'shoot-1, or about shoots at 357 pmol’endosperm' the level of free IAA in these tissues (experimental section I). Further experiments with longer incubation periods showed that [3H]IAA and [3HJOxIAA were further oxidized and Figure 4. 19 The 70 eV mass spectra of the pentafluorobenzyl ester of authentic oxindole-3-acetic acid (a), and that of oxindole-3-acetic acid isolated from Zea mays endosperm (b). This work gave the first precGFSOr-product and MS data for the non- decarboxylation pathway for IAA catabolism in plants. 20 Figure 4». 100 181 75- I46 145 'I. 50- "6 28 369 25- ' 172 89 17 >' -1 77 '6' t 371 (D E, o g. 5 35 5s 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 100 m |8l E < ‘ I46 E‘- a: 754 145 %50- 369 17,28 25- o— 75 95 115 135 155 175 195 215 235 255 275 295 m/z 21 glucosylated at the 7 position of the indole ring to 7- hydroxy-oxindole-3-acetic acid (7-OH-OxIAA) and 7-hydroxy- oxindole-3-acetic acid-7'-O-B-D-glucopyranoside (7-OH-OxIAA- glc) (36) (29). GC-MS, and GC-MS and NMR confirmed the identity of OxIAA, and 7-OH-OxIAA and its glucoside in corn, respectively. In kernels of corn, there were 3.1 and 4.8 1 nmol'plant' of 7-OH-OxIAA, and 7-OH-OxIAA-glc respectively; in shoot tissue there were 62 pmol'plant'1 of 7-OH-OxIAA- glc. The 7-OH-OxIAA—glc was estimated in corn by a novel use of an isotope dilution equation (36). Since radiolabeled 7-OH—OxIAA-glc was not commercially available, chemically synthesized [3HJOXIAA was fed to kernels of corn seedlings. The resulting [3HJ-7-OH-OxIAA-glc synthesized in endosperm and shoots, and the initial specific activity of the [3HJOxIAA added to the corn was used in the isotope dilution equation to calculate the endogenous 7-0H-0xIAA-glc in the tissues. Seven-OH-OxIAA content in corn (29) was measured by synthesizing [13CJ-7-OH-OxIAA and using it as an internal standard in the isotope dilution equation: Ci Y = ( —— ' 1) X Cf where X = amount of labeled compound added, C. = 1 initial Specific radioactivity, Cf = final specific radioactivity, and Y = amount of endogenous compound. The initial Specific radioactivity can be the initial specific radioactivity of the radiolabeled compound added to the tissue (non-radioactive isotopes also can be used with a 22 GC-MS quantitation method), or the initial specific activity of a precursor of the compound to be quantitated. In the former case, the tisSue is killed at the addition of the labeled compound, and the compound purified and the final Specific radioactivity determined; in the later case the labeled compound is incubated with the tissue until measur- able amounts Of the metabolite to be quantitated are synthe- sized, the tissue is then killed and the final specific radioactivity of the metabolite determined. UV, NMR, and MS data have shown that dioxindole-3- acetylaspartic acid (DiOxIAA-asp) and 3-(O-B-glucosyl)- dioxindole-3-acetylaspartic acid (glc-DiOxIAA-asp) are naturally occurring compounds in Vicia faba (61) (62). Radiolabeling feeding studies have shown that Vicia faba catabolizes IAA with carboxyl retention by the following pathway:IAA-->IAA-aSp-->Di0xIAA-asp-->glc-DiOxIAA-asp (63). Free DiOxIAA was not an intermediate in the pathway since [‘“CJIAA was not metabolized to DiOxIAA, and [1"CJDiOxIAA was not metabolized to the DiOxIAA conjugates. A double labeling 3H/mC-IAA-asp experiment showed that IAA-asp was the precursor of the DiOxIAA conjugates. This is the first report of an IAA conjugate (IAA-asp) being an intermediate in an IAA catabolic pathway, rather than a storage form of the hormone IAA. IAA-asp and DiOxIAA-asp were synthesized in both cotyledons and roots, and not transported from the cotyledons to the roots. Interestingly, the glc-DiOxIAA-asp was synthesized only in the cotyledons and was transported 23 with IAA to the roots. The function of the glucosylation and transport of DiOxIAA conjugates from the cotyledons to the roots is unknown. Glc-DiOxIAA-asp was estimated by UV measurements to 1 occur at 8.1 nmole'g fresh weight' in 4-day-old seedlings (61). Epicotyl, cotyledon, and root tissues had 9.1, 8.4, and 9.3 nmol'g fresh weight"1 of glc-DiOxIAA-asp without correction for recovery. Four-chloro-indole-3—acetic was identified by GC—MS in seeds and young leaves of Vicia faba; unchlorinated IAA was below detection < 5 ng'g Fwt"1 (41). Whether 4-chloro-indole-3-acetic acid can be catabolized to the analogous 4-chloro-DiOxIAA conjugates has not been investigated. The occurrence of glc-DiOxIAA-asp in 4 day old seedlings at 3.9 ug’g Ewt"1 (8.1 nmol'g Ewt"1 ) and IAA in young leaves and seeds at < 5 ng'g Fwt'1 suggests that IAA oxidation to DiOxIAA is rapid in broad bean seedlings. Nonhebel et al examined the metabolism of [1uCJIAA in excised dark-grown 3-day-old corn roots, and 5 day-old corn coleoptiles. Excised tissues were incubated in aqueous 1O-5 6 to 10- M IAA, metabolites extracted by methanol, and the methanol extract chromatographed on C18 HPLC. Root segments rapidly metabolized IAA to 11 products following 2 hours of 14 incubation, with only 29 to 34% of the C remaining as IAA (37). A time course experiment showed that within 10 min- utes 4 to 9 IAA metabolites had been produced (38). Only 2 14 to 9% of the C was lost during the 2 h incubation, con- firming that IAA metabolism occurs in corn with carboxyl 24 retention. The metabolism was shown to be independent of incubation and extraction techniques because boiled roots did not catalyze metabolism, and sterilely cultured roots gave the same radioactive profile as non-sterilely cultured roots. However, IAA was more rapidly metabolized by sterilely cultured roots. Corn roots can be separated into vascular stele, and cortex plus epidermis (16). Isolated stelar tissue metabo- lized IAA very slowly, only 1 to 6% in 2 h, while isolated cortex metabolized 91 to 92% of the incorporated IAA (39). It was concluded that the tissue which transports IAA, the stele, slowly metabolizes IAA, while the actively growing tissue, cortex plus epidermis, actively metabolizes IAA. A link between actively growing tissue, and IAA metabolism and regulation of IAA levels is indicated. In 5 day-old corn coleoptiles, 6 to 10 IAA metabolites were isolated following 2 h incubations, with qualitatively the same metabolites as roots (38). However, coleoptiles metabolized IAA at a slower rate; 48 to 51% of the IAA was unmetabolized after 2 h. There were no IAA metabolites in a time course experiment after 10 minutes of incubation, and 2 metabolites after 1 h incubation for excised coleoptiles-- less metabolism than observed with roots. When IAA was fed to coleoptiles in agar blocks, 95% of the 14C was unmetabo- lized IAA in the donor block, 26% of the IAA was unmetabo- lized in the coleoptile tissue, and 98% of the IAA was unmetabolized in the receiver block. Thus, IAA is 25 metabolized in coleoptile tissue, but only IAA is transported basipetally in corn. (Generally hormone transport and ligand binding studies have ignored the possible error of metabolism of IAA in the assays (cf 38).) The study by Nonhebel with excised corn coleoptiles and roots confirms our results with intact seedlings (11) (43) that IAA is metabolized in corn with carboxyl retention. The identity of the IAA metabolites in excised tissues which would include IAA catabolites as well as IAA conjugates was not completed. All of the metabolites were more polar than IAA, again supporting reports that the less polar catabo- lites from peroxidase decarboxylation of IAA were very low in corn. Following the evidence of Reinecke and Bandurski (11), that IAA was catabolized with carboxyl retention to oxindole-3-acetic acid, Nonhebel et al examined the major IAA metabolite of excised root, and coleoptile. 0n C18 HPLC as the free and methyl ester, the metabolite co-chromato- graphed with OxIAA (38). The metabolite which they termed "OxIAA-like" was 21 to 31% of the total 14C in root metabo- lism, and 30 to 33% of the coleoptile metabolism following 2 h incubation periods. Interestingly, the "OxIAA-like" metabolite increased in roots following 10 min, 1 h, and 4 h incubations to the largest metabolite, but decreased to the 4 to 6 largest metabolite following 24 h of incubation. The further metabolism of the "OxIAA-like" metabolite was impli- cated. Klambt (24) reported that developing seedlings of Ribes rubrum, and Aquilegia vulgaris, Triticum coleoptile 26 sections, and Solanum tuberosum tubers formed a glucoside of OxIAA (2-OH-IAA) when fed [14C]IAA. Evidence was based on hydrolysis by B-glucosidase, and acid rearrangement of the indole catabolite to 2-oxo-1,2,3,4 tetrahydroquinoline-4- carboxylic acid. The auxins naphthaleneacetic acid (NAA) and 2,4-D were also reported to be hydroxylated and gluco- sylated by these plants. Further chemical identification of these compounds, and the natural occurrence of the OxIAA glucosides in these plants remains to be demonstrated. The first report of IAA oxidation to OxIAA was in the basidiomycete Hygrophorus conicus (54). The conversion of tryptamine to IAA and then to OxIAA was unique to Hygrophorus conicus of the 12 basidiomycetes tested (69). Oryza sativa, rice, is interesting since it is the only plant known to have OxIAA and DiOxIAA in the same plant (22). Rice was also shown to have the 5-hydroxy analogs of OxIAA and DiOxIAA. The oxindole-3-acetic acids were not isolated from vegetative tissues of rice (only rice bran), nor were radiolabel-feeding studies undertaken to show the precursor-product relationship with IAA. Whether OxIAA can be a precursor for DiOxIAA is also not resolved. Crown gall tumor lines oxidize exogenous IAA by both IAA catabolic pathways (64). Following a 24 h feeding of radiolabeled IAA, at least 33% of the IAA was decarboxylated as shown by base trapping of 1”C 02. Of the remaining IAA in the tissue, 80 to 89% was present in carboxyl-retaining IAA catabolites. The carboxyl retaining catabolites were 27 present in callus and shoot tumor forming lines, as well as in a pith callus tobacco culture. NAA, a synthetic auxin was mostly conjugated to NAA amino acid conjugates under similar growth conditions. The differing degree of stability to catabolism observed with IAA and synthetic auxins explains some of their biological activity differences. The turnover rate of OxIAA in shoots and endosperm -10 1 tissues of corn was estimated to be 1.1 pmol'h shoot” and 7.1 pmol'h'1' 1 endosperm“ (35). The OxIAA turnover value for endosperm tissue was an order of magnitude lower than the IAA turnover value reported by Epstein (11). The IAA turnover study was calculated from an 8 h radiolabled IAA feeding study, while the OxIAA turnover study was calculated from an 18 to 20 h incubation of radiolabled OxIAA following a 6 h pre-incubation period. Interestingly, Nonhebel reports that the initial catabolism of OxIAA in endosperm tissue must have been greater since the initial specific radioactivity decreased by 36 fold during the 6 h pre- incubation period, and only 1.5 fold during the following 18 and 20 h incubation period. Apparently, applied IAA and OxIAA enter into a rapidly turning-over pool and subsequently into a slower turning-over pool. The determination of IAA and OxIAA turnover rates under identical conditions with the same variety of corn would confirm the magnitude of the OxIAA pathway in regulating IAA levels. ‘ A _ rii 28 b. IN VITRO STUDIES The enzymology of the OxIAA and DiOxIAA pathway is much less understood than the peroxidase pathway due to the recent discovery of the OxIAA/DiOxIAA pathway, and the commercial availability of horse radish peroxidase. In Zea mays, enzymatic activity for the oxidation of IAA to OxIAA has been measured in shoot, root, scutellar, and endosperm tissues in the range of 1 to 10 pmol'h'1'mg protein-1 (45) (46). The enzyme is soluble as evidenced by ultracentrifu- gation studies. Enzymatic activity was reduced by 90 per cent when assayed under argon indicating an oxygen require- ment for the reaction, however, 18 O feeding studies are necessary to identify the source of oxygen in the oxindole ring. Enzyme activity was stimulated up to ten fold by preparing enzyme from tissue homogenized with buffer plus non-ionic detergent. A heat-stable component from the Triton X100 prepared enzyme also increased enzyme activity when added to buffer extracted enzyme (Experimental Section II). Cofactors of oxygenation reactions as NADPH, or Fe++, alpha-ketoglutarate, ascorbate, as well as cofactors of peroxidase, are inactive in stimulating OxIAA formation. The identification of the heat-stable, detergent-soluble factor may clarify the mechanism of OxIAA synthesis. The enzymology of IAA oxidation to DiOxIAA has not been investi- gated. 29 The initial attempts by Siehr's laboratory to solubilize the enzyme from the basidiomycete H. conicus resulted in low recoveries (40). Only 10% recovery of enzymatic activity was observed following freeze drying, or sonicating the mycelium. The addition of NADPH, and FADHZ did not increase enzymatic activity (32). These data need to be confirmed since the culture used in these experiments was subsequently lost, and its identity was in question (DJ Siehr, personal communication). An in vitro 5-hydroxylation of IAA and tryptophan was reported for Sedum morganianum (47). However, the products were only identified by spectrofluorimetry and TLC in one solvent system. C. PHYSIOLOGICAL OCCURRENCE OF PATHWAYS Ideally the catabolic route for a plant hormone is initially characterized by radiolabel-feeding studies with physiological applications at physiological concentrations. Subsequently, the endogenous amount of the metabolite is determined as by the isotope dilution method (48) (7). Finally the catabolite's biological activity may be measured. (It should be noted that care must be taken with IAA metabolic studies since IAA may also be non-enzymatical- ly degraded by light, acid, silica gel, etc. (14).) Despite all the research on peroxidase catalyzed decarboxylation of IAA, only a few reports on the isolation and physicochemical identification of peroxidase catabolites have been reported. Indole-3-methanol, and indole-3-carboxylic acid have been 30 identified in pine (51) (55), and indole-3-aldehyde, indole- 3-methanol, and indole-3-carboxylic acid were reported in pea sections fed IAA (31). In pine needles there was 2.3 ng'g fresh weight'1 indole-3-carboxylic acid or about ten per cent of the level of free IAA (51), while in etiolated pine shoots there was 19.7 ng'g fresh weight'1 of indole-3- methanol. Whether indole-3-carboxylic acid is a natural metabolite of IAA is in question since in Brassica its occurrence can be an artifact of glucosinolate breakdown 1u‘C-IAA was not metabolized to indole-3- (55), and in pine carboxylate (50). Three-hydroxymethyloxindole, and 3- methyleneoxindole are unstable compounds, however 3- hydroxymethyloxindole has been reported to occur in pea after feeding labeled IAA to sections (9). Indole-3- methanol glycoside, and indole-3-carboxylic acid were also reported to be catabolites of IAA in wheat sections (27). Generally, catabolites from radiolabel-feeding studies have been identified by thin layer chromatography in several solvent systems; however, substantiation by physicochemical methods is needed. There is no doubt that peroxidase can oxidize IAA in vitro; however, the lack of evidence for naturally occurring catabolites of peroxidase in untreated tissues is a serious weakness in the hypothesis that peroxidase regulates IAA levels in plants. Also, some of the studies measuring [1- 1L‘CJIAA catabolism by 1“CO evolution may have overestimated 2 peroxidase-catalyzed decarboxylation since some of this 31 catabolism might be a cut surface phenomenon (65). For example, 75% of the "IAA oxidase" in pea segments was removed by a 4 minute buffer wash, and "IAA oxidase" activity was proportional to the number of pieces the sections were cut. This evidence along with the fact that over 70% of the total enzyme activity was wall localized may indicate a wound response role for peroxidase oxidation of IAA. Oxindole-3-acetic acid, and dioxindole-3-acetic acid are well documented to be endogenous compounds in three plant species, although the precursor-product relationship has been shown only for broad bean and corn. The occurrence of the carboxyl-retention pathway of IAA catabolism in a monocotyledonous and a dicotyledonous plant indicates that further investigation may identify the pathway in other species as well. Since radiolabeled peroxidase metabolites and OxIAA/DiOxIAA metabolites can be synthesized chemically and enzymatically (27) (43) (60), the natural occurrence of these pathways in other plants may be critically examined by the isotope dilution assay. The comprehensive testing of all the IAA catabolites from the peroxidase, and OxIAA- DiOxIAA pathways in several auxin bioassays is incomplete. However, those catabolites tested are reported inactive in promoting growth in auxin bioassays (43) (53). There are a few reports in the literature of the isola- tion of an "IAA oxidase" without peroxidase activity (53). These enzymes may be a true IAA oxidase catalyzing 32 decarboxylation, the apoprotein of peroxidase, or an enzyme catalyzing IAA to OxIAA or DiOxIAA. The answer to whether IAA is catabolized in vivo by the relatively non-specific peroxidase isozyme system, and/or specifically by another enzyme will clarify how and where IAA catabolism is involved in IAA mediated growth. Attempts have been made to cor- relate peroxidase levels and age of tissue with responsive- ness of tissues to IAA. In some studies there was a positive correlation while other studies showed no correlation (53). The physicochemical measurement of IAA levels in an overproducing or underproducing peroxidase mutant versus the wild type would identify the in vivg role of peroxidase in regulating IAA levels. D. SUMMARY OF IAA CATABOLIC PATHWAYS In summary, there are two pathways of IAA catabolism: the peroxidase catalyzed oxidative decarboxylation of IAA, and the oxidation of IAA without decarboxylation to OxIAA or DiOxIAA. The peroxidase mechanism of oxidation is well understood with purified enzymes; its physiological significance must still be elucidated. The OxIAA-DiOxIAA pathway has been confirmed in only two species; its occurrence in other species must be examined, and the mechanism of enzyme catalysis must be studied further. Future research will identify if IAA catabolism occurs in tissues non-responsive to IAA where IAA is not needed, or in actively growing tissues keeping the hormone concentration 33 limiting following the "growth promoting act". In either case, the elucidation of how and where IAA is catabolized in plants will help clarify how IAA levels are regulated during growth and development. E. REACTION MECHANISM FOR THE OXIDATION OF IAA TO OXIAA The reaction mechanism for the oxidation of IAA to oxindole-3-acetic acid has not been studied for plants. On paper, the conversion of IAA to OxIAA requires the removal of a hydrogen and the addition of an oxygen at the 2 position of the indole ring, and the addition of a hydrogen to the 3 position (fig. 5). Water or molecular oxygen could be the source for oxygen in the oxindole ring. For example, water may add across the pyrole double bond as in the hydratase reactions: aconitase (citrate to isocitrate), fumarase (fumarate to malate), and enoyl COA hydratase (28). The reaction could then be completed by oxidizing the 2- hydroxyoxindole-3-acetic acid to the oxindole. Molecular oxygen could be the source of oxygen for the oxindole ring, as is the case for most oxidation reactions. With a mono- oxygenase reaction, one oxygen of dioxygen is added to the reactant, and the other reduced to water as in phenylalanine hydroxylase (phenylalanine to tyrosine), and tyrosine hydroxylase (tyrosine to DOPA) (20). Also, an inter- molecular dioxygenase reaction could oxidize IAA to OxIAA with one oxygen adding to the indole nucleus and the other to another reactant as in gibberellin 2B-hydroxylase H CH,COOH 34 Figure 5. The ring numbering system for IAA and OxIAA. 35 (gibberellin A1 plus alpha-ketoglutarate to gibberellin A8 plus succinate and C02) (17), and prolyl hydroxylase (proline to hydroxyproline) (1). Interestingly, when the fungus H. conicus was cultured on H2180, there was only a 3% 18 enrichment of C 02 from pyrolyzed OxIAA synthesized by H; conicus mycelium (2). The authors concluded that a hydratase reaction for oxidation of IAA to OxIAA by the fungi was not indicated andithat an oxygenase reaction was 18 O2 incorporation by an oxygenase reaction were deemed unfeas- likely. The convincing .experiments to identify oxygen ible because of the long incubation time required for the fungi to synthesize quatifiableAOxIAA. Other types of oxidation reactions which hypothetically could oxidize IAA to OxIAA are peroxidase, and lipoxygenase (formerly known as lipoxidase in plants). Peroxidase catalyzes the addition of oxygen to the IAA ring via molec- ular oxygen and water (33);; However, the products of this reaction, indole-3-aldehyde: indole-3-methanol, and 3- methyleneoxindole do not retain the carboxyl moiety of IAA as is the case for oxidation to OxIAA. Additionally, oxindole—3-acetic acid is not an intermediate in the decarboxylation reactions of peroxidase (18). Lipoxygenase has been reported to co—oxidize organic molecules as carotene, chlorophyll, and sulfhydryl groups (12) (66) during the oxidation of its natural substrates, unsaturated fatty acids with a cis,cis,1,4-pentadiene structure (linoleic, linolenic, and arachidonic acids). The reaction 36 is thought to be mediated by free radicals or enzyme-bound free radicals resulting from the oxidation of unsaturated fatty acids. A hydroperoxide-dependent hydroxylation of indole to 3- hydroxy-indole (indoxyl) with hydroperoxide reduction to alcohol is catalyzed by pea microsomes (19). The reaction is not specific for indole since phenol, 1-naphthol, and aniline also were oxidized by the pea microsomes system. Of the hydroperoxides tested, linoleic acid hydroperoxide had the lowest Km (0.06 mM) and most physiological pH optimum 7.2. The authors deduced that 02 was not directly involved 18 in the oxidation reaction, since O-linoleic acid hydro- peroxide labeling studies gave an indoxyl product 63% 180- labeled, and since anaerobic conditions had no appreciable effect on indole oxidation. Linoleic acid hydroperoxide was converted mainly to the corresponding alcohol during the reaction. Whether IAA and OxIAA would be oxidized by this system to 3-OH-IAA or OxIAA, and DiOxIAA, respectively, remains to be examined for this system. A summary of possible reaction mechanisms for oxidizing IAA to OxIAA are shown in Figure 6. The elucidation of the mechanism of IAA oxidation to OxIAA would identify cofactors or co—substrates involved in regulating the oxidation reaction. Catabolism of IAA may be a general route to inactivate organic acids or indoles, or a specific enzyme to neutralize a biologically active molecule. OxIAA is inactive in six plant bioassays, Fig. 37 Oxidation reactions which may be model systems for the oxidation of IAA to OxIAA. In reactions 1 to 4 the molecules being oxidized may be cyclic compounds, while in reactions 5 and 6 the initial molecule being oxidized is a fatty acid with a cis,cis,1,4-pentadiene structure. 38 Figure 6. 1) HYDRATASE 2) 3) 4) 5) R1-HC:CH-R2 + 820 --——> R1-H2C-CH(OH)—R2 MONO-OXYGENASE XH R1-CH2-CH2-R2 7 O2 --_-> R,—CH2-CH(OH)-R2 + H2O + x INTERMOLECULAR DIOXYGENASE XH R1-CH2-CH2-R2 + KETOGLUTARATE -E;:i R1-CH2-CH(OH)-R2 + x + SUCCINATE + CO2 PEROXIDASE R1-CH2-CH2-R2 + H202 —-—-> R1-CH2-CH(OH)-R2 7 H2O LIPOXYGENASE (CO-OXIDATION) O 2 1 R,-CH=CH-CH2—CH=CH-R2 -->-->R,_CH=CH-CH=CH-CH(OO >-R2 -—> XH ' R1-CH=CH-CH:CH-CH(OO >-R2 -—> R1-CH:CH-CH=CH-CH(OOH)-R2 ' + X 1 1 X + 02 —-> XOO "PEROXYGENASE" x R,—CH=CH-CH=CH-CH(OOH)-R2 ---> R1-CH=CH-CH=CH-CH(OH)’R2 +X0 39 indicating a role for the reaction in regulating plant hormone level. Several questions have been asked in this present work: 1) is OxIAA a naturally occurring compound in vegetative tissues, 2) in what tissues does the enzyme system which oxidizes IAA to OxIAA occur, and 3) what is the reaction mechanism for this oxidation reaction. The answers to these questions have been investigated by quantitating the levels of OxIAA in corn tissues, and by quantitating and initiating the characterization of the enzyme which oxidizes IAA to OxIAA in vitro. Portions of this literature have been previously reviewed in Plant Hormones and their role in plant growth and development, Peter Davies, ed, Martinus Nijhoff, Dordrecht; In Press 1987, Auxin Synthesis and Metabolism, Dennis M. Reinecke and Robert S. Bandurski. LITERATURE CITED Abbot MT, S Udenfriend 1974 A1pha-ketoglutarate-coupled dioxygenase. In: Molecular mechanisms of oxygen activation. 0 Hayaishi (ed) Academic Press, New York, pp 167-214 Allen W 1969 The incorporation of oxygen-18 into oxindole acetic acid by cells of Hygrophorus conicus. MS thesis. University of Missouri-Rolla Bandurski RS, HM Nonhebel 1984 Auxins. In M Wilkins, ed, Advanced Plant Physiology. Pitman Press, London, pp 1-20 Bandurski RS, A Schulze 1977 Concentration of indole-3- acetic acid and its derivitives in plants. Plant Physiol 60:211-213 Bandurski RS, A Schulze, D Reinecke 1985 An attempt to localize and identify the gravity sensing mechanism of plants. The Physiologist 28:3-111-112 BeMiller JN, W Colilla 1972 Mechanism of corn indole-3- acetic acid oxidase in vitro. Phytochem 11:3393- 3402 Cohen JD, BG Baldi, JP Slovin 1986 13C -[Benzene ring]- indole-3-acetic acid. A new inte nal standard for quantitative mass spectral analysis of indole-3- acetic acid in plants. Plant Physiol 80:14-19 Cohen JD, RS Bandurski 1978 The bound auxins:Protection of indole-3-acetic acid from peroxidase-catalyzed oxidation. Planta 139:203-208 Davies PJ 1972 The fate of exogenously applied indoleacetic acid in light grown stems. Physiol. Plant. 27:262-270 10. Engvild KC, H Egsgaard, E Larson 1980 Determination of 4-chloro-3-indole-3-acetic acid methyl ester in Lathyrus, Vicia and Pisum by gas chromatography- mass spectrometry. Physiol Plant 48:499-503 40 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 41 Epstein E, JD Cohen, RS Bandurski 1980 Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol 65:415-421 Gailliard T 1978 Lipolytic and Lipoxygenase enzymes in plants and their action in wounded tissues. G Kahl, ed, Biochemistry of Wounded Plant Tissues. Walter de Gruyter and Co, New York, pp 155-201 Galston AW, J Bonner, RS Baker 1953 Flavoprotein and peroxidase as components of the indoleacetic acid oxidase system of peas. Arch Biochem Biophys 42:456-470 Galston AW, WS Hillman 1961 The degradation of auxin. In W Ruhland, ed, Hanbuch der Pflanzen Physiologic. Springer-Verlag, Berlin, pp 647-670 Grambow HJ, B Langenbeck-Schwich 1983 The relationship between oxidase activity, peroxidase activity, H O , and phenolic compounds in the degradation of igdgle-3-acetic acid in vitro. Planta 157:131-137 Greenwood MS, JR Hillman, S Shaw, and MB Wilkins 1973 Localization and identification of auxin in roots of Zea mays. Planta 109:369-374 Hedden P, JE Graebe 1982 Cofactor requirements for the soluble oxidases in the metabolism of the C20- gibberellins. J Plant Growth Regul 1:105-116 Hinman RL, J Lang 1965 Peroxidase catalyzed oxidation of indole-3-acetic acid. Biochem 4:144- 157 Ishimaru A, I Yamazaki 1977 Hydroperoxide-dependent hydroxylation involving "H20 -reducible hemoprotein" in microsomes OF pea seeds. JBC 252:6118-6124 Kaufman S, DB Fisher 1974 Pterin-requiring aromatic amino acid hydroxylases. In: Molecular mechanisms of oxygen activation. 0 Hayaishi (ed), Academic Press, New York pp 285-349 Kenten RH 1955 The oxidation of indolyl-3-acetic acid by waxpod bean root sap and peroxidase systems. Biochem. J. 59:110 Kinashi H, Y Suzuki, S Takeuchi, A Kawarada 1976 Possible metabolic intermediates from IAA to B-acid in rice bran. Agric Biol Chem 40:2465-2470 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 42 Klambt HD 1959 Die 2 Hydroxy-Indol-3-essigsaure, ein pflanzliches Indolderivat. Naturwiss 46:649 Klambt HD 1964 2-hydroxyindole-3-acetic acid and similar compounds in seeds and other plant parts. In: Regulateurs Naturels de la Croissance Vegetale, pp235-239, ed JP Nitsch, CNRS, Paris Kogl FA, J Haagen-Smit, H Erxleben 1934 Uber ein neues auxin (heteroauxin) aus harn. Zeit Physiol Chem 228:104—112 Kokkinakis DM, JL Brooks 1979 Hydrogen peroxide— mediated oxidation of indole-3-acetic acid by tomato peroxidase and molecular oxygen. Plant Physiol. 64:220-223 Langenbeck-Schwich B, HJ Grambow 1984 Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments. Physiol. Plant. 61:125-129 Lehninger AL 1977 Biochemistry. Worth Publishers Inc, New York Lewer P, RS Bandurski 1984 Occurrence of 7-hydroxy- oxindole-3-acetic acid in seedlings of Zea mays. Plant Physiol 80(S)95 Magnus V, RS Bandurski, A Schulze 1980 Synthesis of 4,5,6,7 and 2,4,5,6,7 deuterium-labeled indole-3- acetic acid for use in mass spectrometric assays. Plant Physiol 66:775-781 Magnus V, S Iskric, S Kveder 1971 Indole-3-methanol - A metabolite of indole-3-acetic acid in pea seedlings. Planta 97:116-125 Mehta JM 1968 A study of succinic dehydrogenase, indole acetic acid oxidase, nitrate reductase and cytochrome c reductase in Hygrophorus conicus. PhD. University of Missouri-Rolla Nakono M, S Kobayashi, K Sugioka 1982 Peroxidase catalyzed oxidation of indole-3-acetic acid. In: Oxygenases and oxygen metabolism. pp 245-254 Academic Press Nickell LG 1983 IN: Plant Growth Regulating Chemicals. vol 1 and 2, CRC Press, Boca Raton, Florida Nonhebel HM 1986 Measurements of the rates of oxindole- 3-acetic acid turnover, and indole-3-acetic acid oxidation in Zea mays seedlings. J Exp Bot 37:1691- 1697 36. 37. 38. 39. no. ”1. '42. 43. L111. 45. ”6. H70 43 Nonhebel HM, RS Bandurski 1984 Oxidation of indole—3- acetic acid and oxindole-3-acetic acid to 2,3- 1 dihydro-7-hydroxy—2—oxo-1H indole-3-acetic acid-7 - O-B-D-glucopyranoside in Zea mays seedlings. Plant Physiol 76:979-983 Nonhebel HM, A Crozier, JR Hillman 1983 Analysis of [ C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse- phase high performance liquid chromatography. Physiol Plant 57:129-134 Nonhebel HM, JR Hillman, A Crozier, MB Wilkins 1985 Metabolism of [ C]indole-3-acetic acid by coleoptiles of Zea mays L. J Exp Bot 36:99-109 Nonhebel HM, JR Hillman, A Crozier, MB Wilkins 1985 Metabolism of [ C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots. Planta 164:105-108 Patterson B, 1965 Studies on the conversion of indole acetic acid to oxindole acetic acid by Hygrophorus conicus. MS thesis. University of Missouri-Rolla Pless T, M Bottger, P Hedden, J Graebe 1984 Occurrence of 4-Cl-indoleacetic acid in broad beans and correlation of its levels with seed development. Plant Physiol 74:320-323 Rayle DL, RE Cleland 1970 Enhancement of wall loosening and elongation by acid solutions. Plant Physiol 46:250-253 Reinecke DM, RS Bandurski 1981 Metabolic conversion of C-indole-3-acetic acid to C-oxindole-3-acetic acid. Biochem Biophys Res Commun 103:429-433 Reinecke DM, RS Bandurski 1983 Oxindole-3-acetic acid, an indole-3-acetic acid catabolite in Zea mays. Plant Physiol 71:211-213 Reinecke DM, RS Bandurski 1984 Oxidation of indole-3- acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays seedlings. Plant Physiol 75(3):108 Reinecke DM, RS Bandurski 1985 Further characterization of the enzymatic oxidation of indole-3-acetic acid to oxindole-3-acetic acid. Plant Physiol 77(S)3 Reynolds, JD, TD Kimbrough, B Weekley 1983 Evidence for enzymatic 5-hydroxylation of indole-3-acetic acid ”8. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. L121 in vitro by extracts of Sedum morganianum. Z Pflanzenphysiol Bd 112, 465-4700 Rittenberg D, GL Foster 1940 A new procedure for quantitative analysis by isotope dilution with application to the determination of amino acids and fatty acids. J Biol Chem 133:737-744 Sabater F, J Sanchez-Bravo, M Acosta 1983 Effects of enzyme/substrate ratio and of cofactors on the oxidation of indole-3-acetic acid catalyzed by peroxidase. Revista Espanola de Fisiologia 39:169- 174 Sandberg G 1984 Biosynthesis and metabolism of indole-3- ethanol and indole-3-acetic acid by Pinus Sylvestris L. needles. Planta 161:398-403 Sandberg G, E Jensen, A Crozier 1984 Analyses of indole- 3-carboxylic acid in Pinus sylvestris needles. Phytochem. 23:99-102 Schneider EA, F Wightman 1978 Auxins. In: Phytohormones and Related Compounds: A Comprehensive Treatise. DS Letham, PB, Goodwin, TJV Higgins (eds), 1:29-105. Amsterdam: Elsevier/North-Holland. Sembdner G, D Gross, H-W Liebisch, G Schneider 1981 Biosynthesis and metabolism of plant hormones. In: Hormonal Regulation of Development. I. Molecular Aspects of Plant Hormones (Encyclopedia of Plant Physiology Ser: Vol. 9), J MacMillan (ed),pp. 281- 444. Berlin: Springer-Verlag Siehr DJ 1961 The formation of oxindole-3-acetic acid from indoles by a basidiomycete. J Am Chem Soc 83:2401—2402 Sundberg, B, R Sandberg, E Jensen 1985 Identification of indole-3-methanol in etiolated seedlings of Scots Pine (Pinus sylvestris L.). Plant Physiol 77:952- 955 Sundberg B, G Sandberg, and E Jensen 1985 Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris). Physiol Plant 64:438-444 Theologis A, TV Huynh, RW Davis 1985 Rapid induction Of specific mRNAs by auxin in pea epicotyl tissue. J Mol Biol 183:53-68 Thimann, KV 1934 Studies on the growth hormone of 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 45 plants. VI. The distribution of the growth substance. J Gen Phys 18:23-34 Torti G, L Lombardi, LA Manzocchi, F Salamini 1984 Indole—3-acetic acid content in viable defective endosperm mutants Of maize. Tsurumi S, S Wada 1980 Metabolism of indole-3-acetic acid and natural occurrence of dioxindole-3-acetic acid derivatives in Vicia roots. Plant and Cell Physiol 21:1515-1525 Tsurumi S, S Wada 1985 Identification of 3-(0-B- glucosyl)-2-indolone-3-acetylaspartic acid as a new indole-3-acetic acid metabolite in Vicia seedlings. Plant Physiol 79:667-671 Tsurumi S, S Wada 1986 Identification of 3-hydroxy-2- indolone—3-acetylaspartic acid as a new indole-3- acetic acid metabolite in Vicia roots. Plant Cell Physiol 27:559-562 Tsurumi S, S Wada 1986 Dioxindole-3-acetic acid conjugates formation from indole-3-acetylaspartic acid in Vicia seedlings. Plant Cell Physiol 27:1513-1522 Vijayaraghavan SJ, WL Pengelly 1986 Bound auxin metabolism in cultured Crown-gall tissues of tobacco. Plant Physiol 80:315-321 Waldrum JD, E Davies 1981 Subcellular localization of IAA oxidase in peas. Plant Physiol 68:1303-1307 Weber F, W Grosch 1976 CO—oxydation of a carotenoid by the enzyme lipoxygenase: influence on the formation of linoleic acid hydroperoxides. Z Lebensm Unters- Forsch 161:223-230 Went FW, KV Thimann 1978 Phytohormones. Reprinted 1978 Allanheld,Osmun/Universe Books, New York Wightman F, DL Lighty 1982 Indentification of phenylacetic acid as a natural auxin in the Shoots of higher plants. Physiol Plant 55:17-24 Schuytema EC, MP Hargie, I Merits, JR Schenck, DJ Siehr, MS Smith, EL Varner 1966 Isolation, character- ization, and growth of basidiomycetes. Biotech & Bioeng 8:275-286 EXPERIMENTAL I OXINDOLE-3—ACETIC ACID, AN INDOLE-3-ACETIC ACID CATABOLITE IN ZEA MAYS 46 47 ABSTRACT A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays, and that the amount of OxIAA the same has been thus the in both shoot and endosperm tissue is approximately as the amount of free IAA. Oxindole-3-acetic acid reported to be inactive in growth promotion, and rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth. 48 INTRODUCTION Indole-3-acetic acid in the endosperm of germinating seedlings is formed and destroyed at about 96 pmol/h (3). Only 12 pmol/h of this turnover was accounted for by catabolic decarboxylation following application of [1- 1”CJIAA to the endosperm. The major catabolite of IAA, which retains the carboxyl group, has recently been identi- fied as OxIAA by demonstrating that [1-1”C]IAA is catabo- lized to [1-1uC]OxIAA by Z. mays endosperm (13). Most prior studies of IAA catabolism examined the in vitro oxidation of IAA by purified horse radish peroxidase (8) and by tissue homogenates (of. 5,16). Other workers studied the degradation of IAA applied to excised tissues (of. 5,16), and such studies demonstrated that IAA was oxidatively decarboxylated to 3-methyleneoxindole, 3- hydroxymethyloxindole, or indole-3-aldehyde. Recently, Waldrum and Davies (20) showed that much of the IAA decarboxylation in pea epicotyl segments occurred at the cut surface owing to the tissue's cell-wall localized peroxi- dases. Thus, it is possible that the magnitude of the peroxidative route for IAA catabolism has been over emphasized owing to the localization of peroxidase in cell walls (cf. 20) and to the use of in vitro peroxidase assays. 49 In our studies (3) (13), the IAA was applied to the cut endosperm surface of the germinating seedling where cut surfaces of living cells are minimal; and it is possible that this more natural route of application via nutritive tissue reveals the in vivo pathway of IAA catabolism. In this work, we report that OxIAA is an endogenous compound in shoot and endosperm tissue of Z. mays. The levels of OxIAA observed are similar to those of free IAA in these tissues. A previous abstract of these studies has appeared (14). 50 MATERIALS AND METHODS PLANT TISSUE. Corn kernels, Zea mays cv Stowell's Evergreen Sweet Corn (W. Atlee Burpee Co.) were surface sterilized in 1% NaOCl for 10 min, then soaked in running water at 250 C for 16 h. After imbibition, the seeds were grown in moist paper towels at 250 C and 80% RH (3) for an additional 80 h for the endosperm experiments, or were planted in trays of moist vermiculite for an additional 104 h for the shoot experiments. Both the paper towel-grown seedlings and the vermiculite-grown seedlings were harvested at similar stages of growth, 21 i 4.2 cm and 22.0 1 3.9 cm, respectively. A phototropically inactive green safe light was used during necessary manipulations. SYNTHESIS OF LABELED OXIAA. [1-1uCJOxIAA (10 uCi/umol) was synthesized by the method of Hinman and Bauman (7). Purification was with LH-20 lipophilic Sephadex chromatogra- phy using 2-propanol:H 0 (1:1, v/v) for elution. The UV 2 spectrum, LH-20 retention volume, and the HPLC retention time on a C18 Whatman Partisil 10 ODS column (25 X 0.46 cm) as the free acid and as the PFB ester were as previously reported for OxIAA which had been characterized by MS (13). The radiological purity was estimated to be 95% as measured both by chromatography on C18 HPLC eluted with ethanol:H20 51 (1:4, v/v) plus 1% acetic acid (v/v), and by TLC developed with chloroform:methanol:H20 (85:14:1, v/v/v). EXTRACTION OF TISSUE. After harvesting, the tissue was 0 placed onto solid CO and then stored at -20 C until used 2 for extraction. Shoot or endosperm tissues were homogenized in a 4-liter Waring Blendor in sufficient acetone to make the final acetone:H20 concentration 7:3 (v/v). All assays were by an isotope dilution method, and typical experiments 6 are as follows: 2.46 X 10 dpm of 10 uCi/umol OxIAA was added to 394 g of harvested endosperm tissue, and 2.42 X 106 dpm of 10 uCi/umol OxIAA was added to 2.2 kg of harvested shoot tissue. After homogenization, the acetonezHZO extract (7:3, v/v) was evaporated under reduced pressure to an aqueous solution. For the shoot extract, the aqueous phase was partitioned three times with chloroform, and the chloroform phase discarded. The aqueous phase was then acidified to pH 3 and partitioned with ethyl acetate. The ethyl acetate fraction was evaporated to near dryness and chromatographed on a 10-ml bed volume DEAE Sephadex column, washed with ethanol:H 0 (1:1, v/v) and eluted with 2 ethanol:H 0 (1:1, v/v) with a gradient of O to 5% (v/v) 2 acetic acid. Inasmuch as the purity of the samples varied between experiments, additional partitionings were sometimes required prior to DEAE Sephadex chromatography. Tubes containing radioactivity, at the retention volume of OxIAA from the DEAE Sephadex chromatography, were pooled and taken to near dryness. The samples were resuspended in 52 2-propanol:H20 (1:1, v/v) and chromatographed on LH-20 lipophilic Sephadex and eluted with 2-propanol:H 0 (1:1, 2 v/v). OxIAA from endosperm tissue was isolated as previously described (13). The acetone extract from the endosperm contains yellow lipoidal material which was removed by partitioning of the OxIAA into n-butanol followed by chromatography with nonpolar solvents on DEAE cellulose. The samples were then chromatographed on a 5 ml bed volume DEAE Sephadex column followed by an LH-20 column as described for shoot extracts. Both shoot and endosperm samples were then chromatographed on a 25 X 0.46 cm Partisil-10 ODS HPLC column and eluted with ethanol:HZO (1:4, v/v) containing 1% acetic acid (v/v). Radioactive fractions with the retention time of OxIAA were pooled and derivatized to the PFB ester (2). The PFB ester was further purified on C18 HPLC and eluted with ethanol:HZO (43:57, v/v). GC FOR THE DETERMINATION OF SPECIFIC RADIOACTIVITY. The specific radioactivity of the re-isolated OxIAA was determined by a modification of the double standard isotope dilution assay previously described for the measurement of IAA (1) (11). PFB-IAA of known specific radioactivity was used as the second internal standard in the determination of the specific radioactivity of OxIAA. All samples were chromatographed on a 1.8 m x 2 mm i.d., OV-17 column with O the temperature at 200 C for 3 min followed by a 53 temperature program of 200 to 2500 C at 40 C'min-1. The PFB-IAA was purified prior to CC by C18 HPLC using ethanol:HZO (1:1, v/v) as eluant: however, GC indicated a contaminant in the PFB-IAA which would have interfered with the PFB-OxIAA quantitation. Reducing the ethanol concentra- tion from 50 to 43% ethanol (v/v) resolved the PFB-IAA from the contaminant. The plant OxIAA sample contained no IAA detectable by GC (Fig 1); and this was as expected, inasmuch as IAA was separated from OxIAA by LH-20 chromatography, and by both HPLC steps. The PFB-IAA of known specific radio- activity was then mixed with the purified plant PFB-OxIAA to give similar peak areas with a FID. The ratio of peak areas of PFB-IAA to PFB-OxIAA, and the ratio of radioactivity in the peaks of PFB-OxIAA to PFB-IAA were then measured. Peak areas were measured by cutting and weighing photocopies of the GC tracings; and radioactivities were measured by scintillation counting of the collected radioactivity from the column effluent with the FID extinguished, and corrected for counting efficiency. From these measurements, the specific radioactivity of the re-isolated OxIAA was calcu- lated by means of the following equation: IAA peak area OxIAA dpm specific activity ------------- X --------- X of the IAA OxIAA peak area IAA dpm = specific activity of the isolated OxIAA Figure 1. 54 Gas chromatographic elution profile of purified plant IAA. As can be seen, the sample has no contaminants which would interfere with measuring the PFB-IAA internal standard. Under the GC conditions described in the text the retention time of PFB-IAA was two minutes shorter than the retention time of PFB-OxIAA. Figure 1. W '12 136726'23' .- v .4 9 3 1 d 8 I q J I q q d ‘ I 7 6 5 4 3 meOQmmm IOPQMFND 1 ‘ 1-..o 0 MINUTES 56 The rationale of the double standard method for the determination of specific radioactivity has previously been described (1) (11). Briefly, however, the method utilizes a simple proportionality: if a compound of known specific radioactivity is co-injected with a compound of unknown specific radioactivity, then the ratio of radioactivity per peak area of the known compound to that of the unknown may be used to calculate the specific radioactivity of the compound of interest. DETECTOR SENSITIVITY. The detector sensitivity of the FID for PFB-IAA and PFB-OXIAA was determined by separate injections of PFB-IAA and PFB-OXIAA of known specific radioactivities. By measuring the peak area and radio- activity eluting from the column for each standard of known specific radioactivity, the detector responses in peak area per amount of sample injected could be calculated as follows: specific 1/radio- radioactivity X activity X peak = detector sensitivity (nCi/ng) (1/nCi) area (peak area/ng) Because the FID essentially measures the mass of carbon in the compound, the detector sensitivity should be approxi- mately the same for both IAA and OXIAA. The detector sensitivities observed were 5.1 area units/n3 IAA as compared to 5.“ area units/ng OxIAA. The OxIAA values reported for endosperm and shoot tissues were not corrected for detector sensitivity since the sensitivity differences were within experimental error. 57 RESULTS The amount of OxIAA present in the endosperm and shoot tissue was determined from the isotope dilution equation of Rittenberg and Foster (15): Y:[Ci/Cf-1]X The initial specific radioactivity (Ci) of OXIAA was known, the final diluted specific radioactivity (Cf) of OXIAA was measured, and the amount of OxIAA (X) added to the tissue was known. With this information, the equation was solved for the endogenous amount of OXIAA in the tissue (Y). OxIAA was estimated to be 900 and 927 nmol/kg fresh weight in the endosperm, and 220 and 414 nmol/kg fresh weight in the shoot (Table 1) for average values of 914 and 317 nmol/kg fresh weight, respectively. The amount of OxIAA in endosperm tissue on a fresh weight basis was about 3 times higher than that in shoot tissue, while the amount of OXIAA per endosperm was about 7 times higher than the amount of OxIAA per shoot. Epstein et a1 (3) reported amounts of free IAA in dark- grown u-day-old seedlings of 27 pmol/shoot and 308 pmol/endosperm which are similar in magnitude to the levels of OxIAA observed in these experiments (Table II). 58 50362;»: .N ES _ mcosfiucgw soon... 5 soonm>aw63 smut w 2 .o 38 soonm>nw63 smut w ad was .N can _ mcocfiscmsc 833223 E Euommoucobnwfia 58c w and ”£83.. comment—0o wags—B.“ 2: mafia Ems? smut Hum «€on 05 Bob 33328 a?» comma Ba <<~xO .. .<<_xO .«o 3:95 msoaowocao x 63m: 9 Boom <<_xO no 230:8 .N. 5333 258mm :25 .6 5333 2.50% REE ... . Vm Ev o._ on c.~_ we X Nd 50— X c; N 595 vv omm Nd mm _._N mm: X _.v be X Nam _ soonm Now. So and mo m._~ we X Nd Nb— X NN N Euommouum Rm coo and no m._N e2 X m6 he X Nd _ Euommoucm 82%. .8 5.3% N2 «8% $3335: moxbofiz M3 M1 3532:? :5 wow.» l .02 <35 8me .L .x .6 .o “Banana 83:. hosts: =832Q mRSoQ to .3 83.35 Egmknowfiw 35» 82?. .395 .N S EQRQ \o EonV of \o 2333:2me mazcfixcsm V A 2an 59 Table 11. Amount of IAA and OxIAA in Seedlings of Z. mays Tissue OXIAA“ Content IAAh Content pmol/endosperm or shoot Endosperm 357 308 Shoot 49 27 “ Average values from Table I. “ IAA values from Epstein et al. (3). 60 To verify that the peaks measured by GC were PFB-IAA and PFB-OXIAA, the purified plant samples were also analyzed with a Hewlett-Packard 5985 GC-MS using a 3 m x 2 mm i.d. SP2250 column. The PFB-OXIAA plant sample had major 70 eV electron impact fragment ions at 371, 369, 181, 146, 145 as did authentic PFB-OXIAA, and the PFB-IAA internal standard had major fragment ions at 355, 181, 130, 103 as did authentic PFB-IAA (2) (13). 61 DISCUSSION In Z. mays endosperm (13) and in root and shoot segments (12), IAA is rapidly catabolized with retention of the carboxyl group. IAA catabolites which retain the carboxyl group, such as OxIAA, dioxindole-3-acetic acid, and their S-hydroxy derivatives, have been reported in rice bran (9), Z. mays (10), Vicia faba (19), and Hygrophorus conicus (17). We demonstrated a precursor-product relationship between IAA and OxIAA in the endosperm of Z. mays (13) by showing oxidation of [1-1uc11AA to [1-1uCJOxIAA. The present work identifies OxIAA as a naturally occurring compound in both shoot and endosperm tissues. OxIAA occurs at levels similar to that of free IAA in these tissues with 49 pmol OxIAA/shoot and 27 pmol IAA/shoot, and 357 pmol OxIAA/kernel and 308 pmol IAA/kernel. With the knowledge that (a) OXIAA is a naturally occurring compound in Z. mays shoot and endosperm tissues, (b) in vivo oxidation of IAA to OxIAA is a major pathway in endosperm tissue, and (c) OxIAA is generally inactive in growth promotion (6) (9) (18) (21)-although a contrary report has appeared (4), we postulate that the oxidation of IAA to OxIAA plays a role in the regulation of IAA concen- trations and thus in the amount of IAA-mediated growth. 62 Further, there are kinetic reasons why the hormone may be destroyed simultaneously with its action, or shortly after its action, and thus knowledge of the mechanism and site of oxidation of IAA to OxIAA may be important in attaining an improved understanding of how IAA regulates growth. 10. 11. 12. 63 LITERATURE CITED Cohen JD, A Schulze 1981 Double standard isotope dilution assay. I. Quantitative assay of indole-3- acetic acid. Anal Biochem 112:249-257 Epstein E, JD Cohen 1981 Microscale preparation of pentafluorobenzyl esters: electron capture gas chromatographic detection of indole-3-acetic acid from plants. J Chromatogr 209:413-420 Epstein E, JD Cohen, RS Bandurski 1980 Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol 65:415-421 Galston AW, HR Chen 1965 Auxin activity of isatin and oxindole-3-acetic acid. Plant Physiol 40:699-705 Galston AW, WS Hillman 1961 The degradation of auxin. In W Ruhland, ed, Hanbuch der Pflanzen Physiologie. Springer-Verlag, Berlin, pp 647-670 Henderson JHN, CS Patel 1972 Oxindole-3-acetic acid: physical properties and lack of influence on growth. Physiol Plant 27:441-442 Hinman RL, CP Bauman 1964 Reactions of N- bromosuccinimide and indoles. A simple synthesis of 3-bromooxindoles. J Org Chem 29:1206-1215 Hinman RL, J Lang 1965 Peroxidase catalyzed oxidation of indole-3-acetic acid. Biochem 4:144-157 Kinashi H, Y Suzuki, S Takeuchi, A Kawarada 1976 Possible metabolic intermediates from IAA to B-acid in rice bran. Agric Biol Chem 40:2465-2470 Klambt HD 1959 Die 2 Hydroxy-Indol—3-essigsaure, ein pflanzliches Indolderivat. Naturwiss 46:649 Michalczuk L, JR Chisnell 1982 Enzymatic synthesis of 5- 3H-indole-3-acetyl-myo-inositol from 5-3H- tryptophan. J Labelled Compd Radiopharm 19:121-128 Nonhebel HM 1982 The metabolism of indole-3-acetic acid in seedlings of Zea mays L. PhD thesis. University of Glasgow 13. 14. 15. 16. 17. 18. 19. 20. 21. 64 Reinecke DM, RS Bandurski 1981 Metabolic conversion of 14C-indole-3-acetic acid to 14C-oxindole-3-acetic acid. Biochem Biophys Res Commun 103:429-433 Reinecke DM, RS Bandurski 1982 Quantitation of oxindol- 3-y1-acetic acid in Zea mays. Plant Physiol 69:8- 307 Rittenberg D, GL Foster 1940 A new procedure for quantitative analysis by isotope dilution with application to the determination of amino acids and fatty acids. J Biol Chem 133:737-744 Sembdner G, D Gross, H-W Liebisch, G Schneider 1981 Biosynthesis and metabolism of plant hormones. In: Hormonal Regulation of Development. I. Molecular Aspects of Plant Hormones (Encyclopedia of Plant Physiology Ser: Vol. 9), J MacMillan (ed),pp. 281- 444. Berlin: Springer-Verlag Siehr DJ 1961 The formation of oxindole-3-acetic acid from indoles by a basidiomycete. J Am Chem Soc 83:2401-2402 Suzuki Y, H Kinashi, S Takeuchi, A Kawarada 1977 (+)-5- Hydroxy-dioxindole-3-acetic acid, a synergist from rice bran of auxin-induced ethylene production in plant tissue. Phytochem 16:635-637 Tsurumi S, S Wada 1980 Metabolism of indole-3-acetic acid and natural occurrence of dioxindole-3-acetic acid derivatives in Vicia roots. Plant and Cell Physiol 21:1515-1525 Waldrum JD, E Davies 1981 Subcellular localization of IAA oxidase in peas. Plant Physiol 68:1303-1307 Weis JS 1966 Effects of oxindoles on the growth of tobacco tissue cultures. Nature 211:1216-1217 Supported by grants from the Metabolic Biology Section of the National Science Foundation, PCM 7904637, and NASA NAGW-97, 0RD 25796. This is journal article 10586 from the Michigan Agricultural Experiment Station. This article appeared with minor changes in the Journal Plant Physiology 1983 71:211:213. EXPERIMENTAL II ENZYMATIC OXIDATION OF IAA TO OXIAA BY ZEA MAYS 65 66 ABSTRACT Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, endosperm, and scutellar tissues have enzyme activities of 1 to 10 1'mg protein-1. The enzyme is heat labile, is pmol'h' soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymatic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6 to 10 fold. This is the first demonstration of the in vitro enzymatic oxidation of indole- 3-acetic acid to oxindole-3-acetic acid in higher plants. 67 INTRODUCTION 0xindole-3-acetic acid (OxIAA) is a naturally occurring catabolite of indole-3-acetic acid (IAA) in Zea mays (14). The peroxidative decarboxylation pathway is a minor component in corn since feeding [1-1uCJ-IAA to either endosperm of entire seedlings, or root or shoot pieces results in only 1 to 5 per cent decarboxylation per hour of the labeled IAA (2) (13). Twenty-four hour incubations of Zea mays tissues with labeled IAA result in the synthesis of OxIAA, and the further oxidation of OxIAA to 7-hydroxy- oxindole-3-acetic acid (7-0H-0xIAA) and 7-hydroxy-oxindole- 3-acetic acid 71-0-B-D-glucopyranoside (7—OH-0xIAA-glc) (12) (9). Seven-OH-OXIAA and 7-0H-0xIAA-glc are also naturally occurring compounds in corn (12) (9). Recently, Tsurumi and Wada have shown that IAA is conjugated to IAA-aspartate and oxidized to a conjugate of dioxindole—3-acetic acid (3-(O-B-glucosyl)-2-indolone-3- acetylaspartic acid) in the dicot Vicia faba (18) (19). Dioxindole-3-acetic acid (DiOxIAA) is oxidized at the 2 and 3 positions of the indole ring while OxIAA is oxidized at only the 2 position of the ring, both with carboxyl reten- tion. OxIAA and DiOxIAA and their S-hydroxy analogs were also shown to occur naturally in Oryza sativa (rice) bran 68 (7). The occurrence of IAA catabolites which retain their carboxyl moiety in monocots and a dicot may indicate a wider distribution of non-decarboxylating pathways. The in vitro decarboxylation of IAA catalyzed by horse radish peroxidase is well characterized (10). The present work is the first demonstration of an enzyme system which catalyzes oxidation of IAA without decarboxylation. A previous abstract of these studies has appeared (16). 69 MATERIALS AND METHODS PLANT MATERIALS. Zea mays cv Stowell's Evergreen Sweet corn kernels (W. Atlee Burpee Co. Warminster, Pa) were surfaced sterilized in 1% NaOCl for 10 minutes, then soaked in running water at 150 C for 16 h. After imbibition, the kernels were grown in moist paper towels for an additional 80 n at 25° c and 80: relative humidity. Shoot (coleoptile plus primary leaves and mesocotyl), kernel (endosperm plus scutellum), and root tissues were excised and harvested into ice-chilled beakers, using phototropically inactive green light for manipulations. For the sterile-culture experi- ments, kernels were germinated on sterilized moist paper towels in 2.5 x 25 cm culture tubes. ENZYME PREPARATION. Endosperm, shoot, or root tissues were homogenized in 0.05 M phosphate buffer pH 7.0 contain- ing 5% (w/w tissue) insoluble polyvinylpyrrolidone with a tissue to buffer ratio of 2:3 (w/v). Triton X100 0.4% (v/v) was added to the buffer extraction medium where indicated. Following homogenization of plant tissue, the buffer extract was filtered through 2 layers of cheese cloth and centri- fuged at 12,000 x g for ten minutes. The resulting super- natant fluid was dialyzed overnight against 2.5 liters of extraction medium. The dialyzed solution was centrifuged at 70 12,000 x g for 10 minutes; the supernatant fluid was assayed immediately for activity or frozen in liquid N2 for subse- quent assays. Over 80% of the initial enzymatic activity could be recovered following 2 months of storage at -1960 C. ENZYME ASSAY. The standard enzymatic assay included 0.9 ml of enzyme plus 0.1 ml of addendum (metal ions, sulfhydryl reagents, or cofactors of oxidation reactions dissolved in 0.05 M phosphate buffer, see Results). [1-1uCJIAA (57 1, 50 uCi'ml"1 mCi‘mmol' Amersham) diluted with 2-propanol 2:8 v/v was the substrate for the assay. The radiolabeled IAA was 98% radiochemically pure as determined by C18 HPLC. The enzymatic reaction was initiated by the addition of 10 ul of the diluted [1-14CJIAA (1.7 nmole) to the enzymatic mixture, and incubated with shaking (Dubnoff Metabolic Shaking Incubator, Precision Scientific Co.) at 300 C for 1 to 12 h. The reaction was terminated by the addition of acetone to the assay mixture 2:1, v/v. Acid precipitation of the protein was avoided to reduce non-enzymatic breakdown of the acid-labile IAA and OxIAA. Carbon-14 recoveries for the enzyme reaction were determined by mixing a 20 ul aliquot of the enzyme mixture with 200 ul of 0.1% aqueous phosphoric acid, followed by the addition of scintillation cocktail (Safety-solve RPI). Radioactivity was measured on a Beckman LS 7000 scintillation counter. Samples were frozen at -200 C until assayed by HPLC. Samples were centrifuged at 12,000 x g for 10 minutes; the resulting supernatant fluid was evaporated to near dryness with 71 reduced pressure at 350 C. Ethanol, H 20, and concentrated acetic acid were added to the sample to make a final concentration of 20:79:1 (v/v/v), respectively, in 200 ul. A 10 ul aliquot was mixed with scintillation cocktail, and the radioactivity was counted to estimate recovery. The remainder of the sample was chromatographed on a Varian 5000 HPLC, 0.46 x 25 cm 10 um Partisil 10 ODS column (Whatman Inc) with a Co:Pel ODS precolumn (Whatman Inc), and eluted with ethanol:H20:acetic acid (20:79:1 v/v/v, solvent system I) at 1 ml per min. Eluant between 5 and 8.5 minutes was collected in 0.5 ml fractions and radioactivity deter- mined by scintillation counting. OxIAA had a retention time of 7.5 to 7.8 minutes under these conditions, and 7-0H-0xIAA and IAA had retention times of 5.6 and 18 minutes, respec- tively. For more complete radioactive profiles, eluant between 2 and 20 minutes was collected. In later experi- ments, the solvent system was switched at 8 min to 100% ethanol and 2 ml per minute resulting in elution of IAA at 14 min. This method decreased the assay time (baseline resolution between IAA and OxIAA was maintained) and cleaned the column between injections. A channel ratio H# method showed the efficiency of radioactivity counting to be 85- 90%. 14C The amount of OxIAA formed was estimated by the recovery and the specific radioactivity of IAA using the following equation: 72 14 14 . . . HPLC CINITIAL initial nmol IAA - x -------- x — ------------- - T4 T4 CHPLC C IAAINITIAL 1n _ 1a CINITIAL ‘ C IAA C OXIAA nmol OxIAA synthesized. Since INITIAL’ the equation can be reduced to: 1A . . . C OxIAAHPLC initial nmole OxIAA ---------------------- X nmole IAA = synthesized THC /THC incubation recoverya 1n _1u HPLC- CHPchTotal radiolabel applied to HPLC; incubation recovery=% recovery of radiolabel at where C OxIAA 14 C OxIAA isolated following HPLC; 14C termination of the enzymatic reaction (athis factor corrects for any decarboxylation and thus under- 1n estimation of C recoveries following the enzymatic reaction); initial nmole IAA=nmole IAA added to the 1M ' enzymatic reaction; and C IAAinitial and 14 . . . Cinitial-radiolabeled IAA added to the enzymatic reaction. For example, if [1”]OXIAA at HPLC was 10,000 DPM and 14 C at HPLC (corrected for incubation loses) was 100,000 DPM (or OxIAA=10% of total), and 1 nmole of IAA was added to the reaction mixture, then 0.1 nmole of OxIAA was synthesized and 0.9 nmole of IAA remains. The equation is valid since IAA destruction and OxIAA synthesis occur 1:1, and since the recoveries of IAA and OxIAA are essentially identical following HPLC. VALIDATION OF ENZYME ASSAY. The enzyme assay was validated by a reverse isotope dilution assay (3). Unlabeled OxIAA was added to the enzyme mixture following termination of the reaction by acetone. The specific radio- 73 activity of the OxIAA was determined by measuring the radio- activity in the OxIAA HPLC peak vs the area of the 254 nm absorbance of the peak. The 254 nm absorbance area was integrated by an an IBM 9000 computer and compared to an OxIAA standard curve (absorbance vs OxIAA amount). Results from the two quantitation methods were identical, and the radioactivity recovery method (see preceding section) was routinely used to quantitatively estimate OxIAA synthesized. To determine whether 7-0H-OxIAA was synthesized by the in vitro system the following solvent system II (9) was used: 10% A plus 90% B from 0 to 5 min, followed by a gradient to 30% A plus 70% B from 5 to 20 min (A=ethanol plus 0.1% acetic acid, and B=H 0 plus 1% acetic acid). 2 Biological activity of OxIAA was measured by a Zea mays mesocotyl straight growth assay according to the method of Nitsch (11). Mesocotyls were incubated in the dark for 20 h in pH 5.0 citrate-phosphate buffer plus 20 mg/L of chlor- amphenicol, 2% sucrose (w/v), and IAA or OxIAA. Mesocotyl length was measured to the nearest 0.1 mm using a dissecting microscope. Gas chromatography of the putative pentafluorobenzyl ester of IAA (15) was performed on a Varian 3700 gas chromatograph with a 2 mm ID x 1.8 meter 0V-17 column and with nitrogen as the carrier gas. The oxygen requirement for the reaction was examined by 10 min vacuum evacuation of 10 ml Thunberg tubes containing the complete reaction mixture, followed by a 30 second argon 74 or air flushing. The evacuation and flushing was repeated 3 OC. The Thunberg tubes were incubated at 300C times at 0 for 4 h and the reaction mixture assayed for OxIAA formation as previously described. The sedimentation characteristics of the enzyme were examined by centrifugation at 100,000 X g for 1 h on a Spinco model L centrifuge with a 50Ti rotor. The prepara- tion was assayed prior to centrifugation, and the superna- tant fluid and resuspended pellet were assayed following centrifugation. 75 RESULTS Since the enzymatic assay is an end-point analysis the linearity over time and protein concentration was deter- mined. The enzymatic reaction was a linear function of time up to 4 hours, and slowed between 4 to 6 hours (Fig. 1). The leveling off of activity could not be accounted for by the depletion of substrate IAA. Enzymatic activity for endosperm plus scutellum was linear over the protein concentration range 0.1 to 2.4 mg protein'ml'1, and linear from 0.1 to 1 mg protein'ml-1 for vegetative mesocotyl tissue. Standard incubation periods were 4 hours for detergent extracted enzyme and buffer extracted enzyme, with 1 to 2 mg protein'ml'1. The pH optimum for the enzyme assay was 7.0. Enzymatic activity was destroyed after 5 to 10 00. minute at 100 The enzymatic activity from sterilely cultured corn seedlings and non-aseptically cultured corn was 1.1 pmol'h' 1- 1'mg protein'-1 respectively. mg protein"1 and 1.0 pmol'h' Fungal contamination was not present in the aseptically grown corn as shown by culturing of corn and media from the aseptically cultured corn on potato dextrose agar. Thus, the oxidation rate of IAA by non-aseptically grown corn was not influenced by microbial contamination, and corn kernels 76 *1 o I I I I I I I O 2 4 6 8 1O 12 time(h) Fig. 1. Linearity of enzymatic activity as a function of incubation time. Enzyme was prepared by tissue homogenization with 0.05 M phosphate buffer plus Triton X100 as described in Materials and Methods. 77 sterilized with 1% NaOCl and grown non-sterilely were used routinely for enzyme preparations. Triton prepared enzyme from shoot, root, and endosperm 1 1 tissues had 1 to 10 pmol‘h' 'mg protein" of enzymatic activity. The highest activity was from endosperm tissue at 1'mg protein-1, while shoot and root tissues 1. 6 to 10 pmol'h' mg protein-1. Vegetative tissues had 14 had 3 to 5 pmol°h' the lowest recovery of radioactive C following the assay with 30 to 60% recovery. For vegetative tissues, the lower rate of oxidation of IAA to OxIAA may be partly explained by the lower availability of substrate IAA due to the competing peroxidase reaction. Peroxidase activity has been reported in corn tissues, but its in vivo role in oxidizing IAA has been shown to be minimal (2) (13). The chromatographic properties of the product of IAA's enzymatic oxidation are shown in Table 1. Chromatography as both the free acid, pentafluorobenzyl ester, and acid ring- expanded OxIAA (quinolone-4-carboxylic acid (6) ) were similar for both authentic and enzymatically synthesized OxIAA. OxIAA has been shown to be a naturally occurring compound by GC-MS in Zea mays tissues (15) and chromato- graphic evidence also supports the in vitro oxidation of IAA to OxIAA. The further oxidation of OxIAA to 7-OH-OxIAA or 7-0H-OxIAA-glucoside was not observed in the in vitro system. A polar catabolite of IAA with a similar retention time to 7-0H-0xIAA with HPLC solvent system I was often synthesized along with OxIAA, but it was separated from 78 Table 1. Authentic OxIAA and enzymatically synthesized putative OxIAA have the same chromatographic properties as evidenced by HPLC and GLC. .RETENTION TIME ENZYME CHROMATOGRAPHY OXIAA PRODUCT HPLC FREE ACID 7.5 7.5 HPLC PFB ESTER ll.8 12.0 GLC PFB ESTER 17.3 17.5 METHYL ESTER HPLC QUINOLINE 5-1 5-0 79 7-OH-OxIAA under C18 HPLC solvent system II (see Materials and Methods). The unknown had a retention volume of 20 to 21 ml and the 7-0H-OxIAA 24 ml with HPLC solvent system II; the putative and authentic OxIAA had retention volumes of 30-31 m1. Flushing and vacuum evacuation of air from Thunberg tubes decreased enzymatic activity by 1.4 fold as compared to the un-manipulated air control. Flushing the Thunberg tubes 3 times with argondecreased enzymatic activity 9 times vs the air flushed control (Fig: 2) showing oxygen was required for optimal enzymatic activity. However, demon- stration of oxygen incorpOration into the indole ring awaits 1802 experiments. A '1 . When enzyme preparations were centrifuged at 100,000 x g for 1 h, both shoot and endosperm preparations retained most of the activity in the supernatant fluid, 90 and 87% respectively (Table 2). Minor activity remained in the unwashed pellet. Sedimentation characteristics of the enzyme preparation resembled a soluble enzyme and not a microsomal enzyme. The results were similar whether the enzyme was prepared with or without Triton X100. Inorganic ions and cofactors of oxygenase reactions were tested for enhancement of enzymatic activity (Table 3, 4). Ca++, and Fe++ were not stimulatory at 50 uM concentrations, while Zn++ at 5 uM was inhibitory by 15 to 20%. Mercaptoethanol and dithiothreitol were inhibitory to enzymatic activity by 80 to 100% when added at 5 to 50 mM. Fig. 2. pmol-h'l-mg protein'1 80 A A 0 M 0| 0) o. ' 0 Control Ar Air Enzymatic activity following evacuation and gas flushing of Thunberg tubes. Ultrapure argon (99.995%, Matheson) was used in these experiments. Preliminary experiments with 99.9% nitrogen showed inhibition of activity up to 4 fold. 81 Table 2. The sedimentation characteristics of shoot and endosperm enzyme preparations. Treatment Endosperm Shoot pMol'h'1 homogenate 44 25 pellet 100,000g 47 35 supernatant 82 and H202, decreased recovery from the enzyme reaction by over 50%, without 14 ++ Cofactors of peroxidase, Mn C increasing the amount of OxIAA synthesized. Peroxidase plus + Mn and H O decarboxylate IAA to hydroxymethyloxindole, 2 2 indole-3-aldehyde, etc. but do not decarboxylate OxIAA (5). The oxidation of IAA to OxIAA is thus a separate pathway from the well studied in vitro oxidative decarboxylation of IAA by peroxidase. Since one oxygen atom is added to the IAA molecule to form oxindole-3-acetic acid, a mixed function oxygenase reaction was indicated. For such a reaction, a reductant would be required since one oxygen would be incorporated into the indole ring and the other oxygen reduced to water. However NADPH at 0.67 mM or at 1.2 mM was not stimulatory to the enzyme's activity. NADPH plus pterin (6,7dimethyl- 5,6,7,8tetra-hydropterine Sigma), 1.2 mM and 0.5 mM respectively, were also not active. At low concentrations (10 uM), FAD was not stimulatory to enzymatic activity. Fe++, alpha-ketoglutarate, and ascorbic acid are co- substrates for the oxidation of gibberellins (4) by a intermolecular dioxygenase reaction by co-oxidation of alpha-ketoglutarate to succinate and 002. But oxidation of IAA to OxIAA was not increased by Fe++, alpha-ketoglutarate, ascorbate at 1 mM, 1 mM, and 5 mM concentrations respec- tively. The substitution of NADPH for ascorbate was also not stimulatory. Thus, none of the most likely types of oxidation reactions, peroxidase, mixed function oxygenase or 83 Table 3. Effect of inorganic ions, and sulfhydryl reagents on the enzymatic oxidation of IAA to OxIAA with endosperm enzyme preparations and Triton X100 endosperm enzyme preparations. Treatment endosperm§ endosperm” (Triton) Fe++ 200 uM 109 500 uM 112 Ca++ so uM 100 25 uM 72 Cu+ nd -- 20 uM 101 Zn++ 5 uM 79 nd -- Mn++ 50 uM 81 100 uM 90a Mercaptoethanol 5 mM 1 5 mM 0 Dithiothreitol nd -- 5 mM 0 § % of control, a plus 17 mM H o 2 2, ndznot determined 84 Table 4. Effect of cofactors and co-substrates of oxygenases and peroxidase reactions on the oxidation of IAA to OxIAA by endosperm enzyme preparations. Treatment Tissue endosperm (Triton X100) 2,4 dichlorophenol 5 uM 87 2,4 dichlorophenol 5 uM + Mn+ 5 uM 97 Fe++ 50 mM + 500 uM Pterin 93 endosperm Pterin 500 uM + 1.2 mM NADPH 42 FAD 1o uM + 1.2 mM NADPH 38 Fe++ 1 mM + 1.2 mM NADPH + 21 ketoglutarate 1 mM Fe++ 1 mM + 5.7 mM ascorbate + 0 ketoglutarate 1 mM Mn++ 0.1 mM + 17 mM H202 90 NADPH 0.7 mM 90 % of control 85 intermolecular dioxygenase, were involved in the oxidation of IAA to OxIAA since cofactors and co-substrates of these reactions were ineffective. To increase recovery of the enzymatic activity during extraction, the non-ionic detergent Triton X100 was added to the homogenization medium. Following preparation with and without 0.4% Triton X100, the enzyme was dialyzed overnight against 0.05 M phosphate buffer, and then assayed. Triton X100 addition to the homogenate prior to the enzyme assay had no effect on enzymatic activity, but homogenization of tissue with buffer plus Triton X100 enhanced enzymatic activity by up to 10 fold. If Triton X100 was added to the homogenate prior to dialysis (absent from the tissue homogenization medium), activity was increased 2 fold without an increase in protein recovery. These experiments indicated that the Triton effect could be due to the preferential extraction of a lipid-soluble enzyme, or of the extraction of a lipid soluble cofactor or co-substrate. The total amount of protein recovered by buffer with and without Triton X100 was similar, so the possible presence of a cofactor was studied by adding boiled Triton prepared enzyme to enzyme (extracted without Triton X100). The addition of the boiled Triton extract increased enzyme activity up to 6 fold (Fig. 3). The addition of boiled enzyme (without Triton X100 preparation) was ineffective in stimulating enzymatic activity. These results indicated that a heat- stable lipid-soluble factor was extracted by Triton X100 and 86 Fig. 3. Stimulation of enzymatic activity by Triton X100 preparation of enzyme, and by the addition of the Triton X100 heat-stable factor to enzyme prepared without detergent. \mm fl. 0mm \\ 2 SW... 88 was responsible for the detergent enhancement of enzymatic activity. OxIAA has been previously shown to be inactive in stimulating plant growth in 5 bioassays (cf 4), although the initial report for pea stem sections showed stimulation. 8 Solutions of 10' to 10'3 M OxIAA and IAA were assayed in a corn mesocotyl bioassay. OxIAA was inactive from 10"8 to 10'” M, while IAA was stimulatory over the same concentra- tion range with maximum activity at 10"7 M (table 5). These results support the view that the oxidation of IAA to OxIAA results in loss of biological activity for the molecule. 89 Table 5. The biological activity of IAA and OxIAA (10‘7 to 10'3 M) in the enhancement of dark-grown corn mesocotyls. The initial lengths of the mesocotyl sections were 4.6:0.1 mm, cut 2 mm below the coleoptile node. Eleven to 13 sections were assayed in each treatment, and data is expressed as treatment length minus initial length. M IAA OxIAA o 1.34:0.39 1.34:0.39 10'7 2.95:1.05 1.34:0.32 1o"6 2.58:0.67 1.59:0.62 10‘5 2.44:0.78 1.48:0.34 To”l 2.36:0.64 1.48:0.42 10'3 1.77:0.36 1.92:0.42 90 DISCUSSION An enzyme system which will oxidize IAA at the 2 position of the indole ring while retaining the carboxyl side chain has been partially characterized in enzyme preparations of corn. This is a novel enzyme system discrete from the peroxidative oxidation of IAA since peroxidase cofactors do not stimulate formation of OxIAA. OxIAA was shown not to be an intermediate in horse radish peroxidase's decarboxylation of IAA (5). The reaction is not a typical mixed function oxygenase reaction in that the enzyme is soluble, and is not stimu- lated by cofactors of mixed function oxygenases. The intermolecular dioxygenases are soluble enzymes as in the oxidation of GA aldehyde dioxygenase, but cofactors of 29 this reaction do not stimulate IAA oxidation. The enzyme requires oxygen for optimum activity. In 180 peroxidase's oxidation of IAA to 3-methyleneoxindole, 2 18 and H2 2 oxygen in the oxindole ring (10). The source of the oxygen 0 experiments showed that H 0 is the source of in the oxindole nucleus of oxindole-3-acetic acid remains to be identified by heavy isotope experiments. The reaction's stimulation by Triton X100 preparation of the enzyme, or by the addition of boiled Triton X100 91 prepared enzyme indicates involvement of a heat-stable, lipid-soluble component in the reaction. The existence of a lipid-soluble, heat-stable component is currently being investigated for this novel reaction. The extent of peroxidase's involvement in catabolism of IAA needs to be re-investigated. In corn, decarboxylation of IAA was only 5 to 12 per cent of the turnover for intact tissues, while isolated corn peroxidase readily catalyzed the oxidation of IAA to 7 major decarboxylated products (1). In pea, peroxidase activity was mainly a cut surface phenomenon which could largely be washed away, and was pro- portional to the number of pieces into which the tissue was cut (20). In Pinus sylvestris, Scots pine, an in vitro system rapidly metabolized IAA to indole-3-methanol and 4 other decarboxylated metabolites. However, when IAA was fed to Scots pine protoplasts, IAA was more slowly metabolized to 2 compounds: an unidentified carboxyl-retaining catabo- lite and indole-3-methanol (minor catabolite) (17). The non-decarboxylation pathways observed in the dicot Vicia faba and the monocot Zea mays (implicated in Oryza sativa, Ribes rubrum, Brassica rapa, and Pinus sylvestris (7) (8) (17)) warrant the re-examination of the route(s) of IAA catabolism in plants, and suggest that the decarboxylation pathway may have been overestimated by the experimental conditions utilized. In corn, IAA is oxidized to OxIAA; OxIAA may be further oxidized to 7-OH-OxIAA and 7-0H-OxIAA-glc. The 92 first enzyme in this pathway has been partially character- ized. Since catabolic oxidation of IAA to OxIAA is the first reaction in the catabolic pathway, and is apparently irreversible (it is inactive in bioassays including corn) it may have an important role in regulating the steady state level of IAA during IAA-mediated growth. 93 LITERATURE CITED 1. BeMiller JN, W Colilla 1972 Mechanism of corn indole-3- acetic acid oxidase in vitro. Phytochem 11:3393- 3402 2. Epstein E, JD Cohen, RS Bandurski 1980 Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol 65:415-421 3. Hall PL, RS Bandurski 1978 Movement of indole—3-acetic acid and tryptophan-derived indole-3-acetic acid from the endosperm to the shoot of Zea mays L. Plant Physiol 61:425-429 4. Hedden P, JE Graebe 1982 Cofactor requirements for the soluble oxidases in the metabolism of the C20- gibberellins. J Plant Growth Regul 1:105-116 5. Hinman RL, J Lang 1965 Peroxidase catalyzed oxidation of indole-3-acetic acid. Biochem 4:144-157 6. Julian PL, HC Printy, R Ketcham, R Doone 1953 Studies in the indole series XIV oxindole-3-acetic acid. J Am Chem Soc 75:5305-5309 7. Kinashi H, Y Suzuki, S Takeuchi, A Kawarada 1976 Possible metabolic intermediates from IAA to B-acid in rice bran. Agric Biol Chem 40:2465-2470 8. Klambt HD 1959 Die 2 Hydroxy-Indol-3-essigsaure, ein pflanzliches Indolderivat. Naturwiss 46:649 9. Lewer P, RS Bandurski 1984 Occurrence of 7-hydroxy- oxindole-3-acetic acid in seedlings of Zea mays. Plant Physiol 80(S)95 10. Nakono M, S Kobayashi, K Sugioka 1982 Peroxidase catalyzed oxidation of indole-3-acetic acid. In: Oxygenases and oxygen metabolism. pp 245-254 Academic Press 11. Nitsch JP, C Nitsch 1956 Studies on the growth of coleoptile and first internode sections. A new, sensitive, straight-growth test for auxins. Plant Physiol 31:94-111 12. 13. 14. 15. 16. 17. 18. 19. 20. 94 Nonhebel HM, RS Bandurski 1984 Oxidation of indole-3- acetic acid and oxindole-3-acetic acid to 2,3- l dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7 - O-B-D-glucopyranoside in Zea mays seedlings. Plant Physiol 76:979-983 Nonhebel HM, A Crozier, JR Hillman 1983 Analysis of [ C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse- phase high performance liquid chromatography. Physiol Plant 57:129-134 Reinecke DM, RS Bandurski 1981 Metabolic conversion of C-indole-3-acetic acid to C-oxindole-3—acetic acid. Biochem Biophys Res Commun 103:429-433 Reinecke DM, RS Bandurski 1983 Oxindole-3-acetic acid, an indole-3-acetic acid catabolite in Zea mays. Plant Physiol 71:211-213 Reinecke DM, RS Bandurski 1985 Further characterization of the enzymatic oxidation of indole-3- acetic acid to oxindole-3-acetic acid. Plant Physiol 77(S):3 Sundberg B, G Sandberg, and E Jensen 1985 Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris). Physiol Plant 64:438-444 Tsurumi S, S Wada 1985 Identification of 3-(0-B- glucosyl)-2-indolone-3-acetylaspartic acid as a new indole-3-acetic acid metabolite in Vicia seedlings. Plant Physiol 79:667-671 Tsurumi S, S Wada 1986 Dioxindole-3-acetic acid conjugates formation from indole-3-acetylaspartic acid in Vicia seedlings. Plant and Cell Physiol 27:1513-1522 Waldrum JD, E Davies 1981 Subcellular localization of IAA oxidase in peas. Plant Physiol 68:1303-1307 EXPERIMENTAL III STIMULATION OF THE OXIDATION OF INDOLE-3-ACETIC ACID TO 0 OXINDOLE-3-ACETIC ACID BY A LIPID FACTOR FROM ZEA MAYS 95 96 ABSTRACT The plant hormone indole-3-acetic acid is oxidized to oxindole-3-acetic acid by a Zea mays enzyme preparation. Enzymatic activity is enhanced by 3 Triton X100 extractable, heat-stable factor. The corn factor was partially purified and characterized from endosperm tissue, and was found to have chromatographic properties similar to that of an unsaturated fatty acid. The stimulatory effect of the corn factor can be replaced by the unsaturated fatty acids linoleic, linolenic, and arachidonic acids. A model system has been constructed for the oxidation of indole-3-acetic acid to oxindole-3-acetic acid using soybean lipoxygenase plus linoleic acid. Lipoxygenase and the corn enzyme have the same specificity for unsaturated fatty acids, and are inhibited by butylated hydroxytoluene, cyanide, and sulfhydryl reagents. However, the Zea mays enzyme does not co-fractionate with Zea mays lipoxygenase suggesting a unique indole-3-acetic acid oxidase system. 97 INTRODUCTION Corn seedlings and corn enzyme preparations (11)(9)(10) (14) oxidize indole-3-acetic acid (IAA)2 with carboxyl retention to oxindole-3-acetic acid (OxIAA). Corn endosperm and vegetative tissues further oxidize OxIAA to 7-hydroxy- oxindole-3-acetic acid (7-OH-0xIAA) and 7-hydroxy-oxindole- 3-acetic acid 7'-O-B-D-glucopyranoside (7-0H-OxIAA-glc) (8) (6). The mechanism of oxidation of IAA to OxIAA is of interest being the first step in the IAA catabolic pathway, and thus the reaction may play a regulatory role in the control of IAA levels, and in IAA mediated growth. The enzyme catalyzing the oxidation of IAA was heat- labile and soluble, and the reaction required oxygen (14). The addition of detergent to the tissue homogenization medium enhanced the enzymatic oxidation of IAA to OxIAA up to 10 fold. Cofactors of peroxidase, mixed function oxygenase, and intermolecular dioxygenase did not stimulate the reaction. The detergent stimulation of enzymatic activity was not caused by enhanced protein extraction, but was due to a heat-stable, lipid-soluble component of corn. The chromatographic properties of the lipid factor, and the involvement of fatty acids in IAA oxidation are examined in 98 the present work. A previous abstract of this work has appeared (13). 99 MATERIALS AND METHODS PLANT MATERIALS. Zea mays cv Stowell's Evergreen Sweet corn kernels (W. Atlee Burpee Co Warminster, Pa) were germ- inated 4 days in moist paper towels as previously described (12). Endosperm (plus scutellum), or scutellum tissues of 4-day old seedlings were harvested into beakers chilled to 00 C. Phototropically inactive green light was used for illumination during manipulations. ENZYME PREPARATIONS. Enzyme preparations from endo- sperm tissue have been described (14). Briefly, tissue was homogenized with an Omni mixer using 0.05 M phosphate buffer pH 7.0 containing 5% (w/w tissue) polyvinylpyrrolidone (PVP), and 0.4% (v/v) Triton X100 (where indicated) with a tissue to buffer ratio of 2:3 (w/v). The extract was filtered through two layers of cheese cloth, and centrifuged 10 min at 12,000 x g. Following dialysis overnight in 2.5 l of 0.05 M phosphate buffer, the sample was centrifuged at 12,000 x g. The supernatant fluid was frozen in liquid nitrogen for enzyme assays or for extraction of the lipid factor. For ammonium sulfate fractionation experiments, 4-day old scutellum tissue was harvested from the endosperm and frozen with liquid nitrogen. The frozen tissue was ground 100 with a mortar and pestle, and extracted with 0.05 M phos- phate buffer (1:4, w/v) plus 5% (w/w) PVP for 30 min at 00 C. The extract was filtered through 2 layers of cheese cloth, then centrifuged at 8700 x g for 15 min and 18,000 x g for 15 min. The supernatant fluid was fractionated with ammonium sulfate (0 to 42%, 42 to 53%, and 53 to 80%, or 0 to 30%, 30 to 42%, 42 to 60%, and 60 to 80%) with slow stirring at 00 C for 30 min. After centrifugation at 12,000 x g for 10 min, the pellet was resuspended in extraction buffer (minus PVP). The resuspended protein was dialyzed overnight against 1 liter of buffer with one change of buffer. The dialyzed protein was centrifuged at 12,000 x g for 10 min, and the supernatant fluids were assayed for enzymatic activity. EXTRACTION OF THE LIPID FACTOR. Lipid factor was isolated from Triton X100 enzyme preparations by boiling the enzyme for 10 min, followed by centrifugation at 12,000 x 10 min. The deproteinized sample was assayed for activity by adding 0.1 to 0.5 ml of the sample to endosperm enzyme (prepared without detergent) for a final volume of 1 ml. Enzymatic activity was measured as described in the follow- ing ENZYME ASSAY section. Enzyme diluted with buffer plus 0.4% Triton X100 was assayed for comparison. The heat- stable lipid factor was further fractionated by chloroform partitioning, DEAE Sephadex acetate, SP Sephadex, and C18 1 HPLC (1 ml’min' on a 0.46x25 cm column). The lipid factor 101 was eluted from a DEAE Sephadex column with ethanol:H 0 1:1 2 (v/v) with a 0 to 5% acetic acid gradient (200 m1). ENZYME ASSAYS. The enzyme assay was performed by O incubating the enzyme plus additions for 1 or 4 h at 30 C on a Dubnoff metabolic shaker. [1-1uCJIAA (58 mCi'mmol'1, 50 uCi'ml'1 Amersham) diluted 2:8 (v/v) with isopropanol was the substrate for the assay. The enzyme assay was initiated by the addition of 10 ul (1.7 nmol) of the diluted [11MCJIAA to 1 ml enzyme preparation. The reaction was terminated by the addition of acetone to the assay medium 2:1 (v/v). The synthesis of radiolabeled OxIAA was measured by a C18 HPLC- scintillation counting method (14) which separated radio- labeled OxIAA from the substrate IAA. Lipoxygenase was assayed by measuring the 234 nm absorbance increase of fatty acid hydroperoxides (1) on a Cary 15 spectrophotometer. The substrate for the assay was 10 mM sodium linoleate dissolved in 0.28% Tween 20 (w/v), mixed according to the method of Axelrod (1). The reaction was initiated by the addition of ten to 50 ul of soybean lipoxygenase (or corn enzyme) to a diluted sodium linoleate solution (10 to 20 ul sodium linoleate diluted to a final volume of 1.2 ml with 0.05 M phosphate buffer pH 7). A 300 to 400 sec time interval during the linear portion of the reaction curve was used to estimate lipoxygenase activity. Fatty acid hydroperoxide isomerase was assayed by measuring the 234 nm absorbance decrease in fatty acid hydroperoxides (3). The isomerase substrate, fatty acid 102 hydroperoxide, was synthesized by soybean lipoxygenase (I) oxidation of linoleic acid in a 0.2 M sodium borate buffer pH 9.0. The isomerase assay was initiated by the addition of five to 10 ul of corn enzyme preparation to the diluted linoleic acid hydroperoxide solution (70 ul of hydroperoxide solution diluted to a final volume of 1.19 ml with 0.05 M phosphate buffer). Protein determination was by the Bradford assay (BioRad) using bovine gamma globulin for standardization. FATTY ACIDS. Fatty acids (linoleic, linolenic, stearic, and palmitic acids, etc., Sigma Chemical Co) were stored under nitrogen at -200 C until assayed. For assays, fatty acids were dissolved in 0.4% Triton X100 sparged with nitrogen. One hundred ul of appropriately diluted fatty acid solution were added to 900 ul of corn enzyme or soybean lipoxygenase. Lipoxygenase-catalyzed oxidation of IAA was measured following a 1 h incubation period with 0.5 to 1 1 ug'ml' of protein since the reaction was a linear function of time for 2 h and of protein concentration between 0.35 and 1.1 ug'ml‘1. Boiled enzyme controls were assayed with each fatty acid concentration, and the non-enzymatic activ- ity subtracted from the enzyme assays. Such controls never exceeded 2% and 23% of the enzyme-catalyzed reaction rate for rapid and slow rates, respectively. 103 RESULTS The Triton X100 enhancement of enzymatic activity is due to a lipid-soluble, heat-stable component of corn tissue (14). The lipid factor was retained by an anion exchanger as the free acid and the lipid factor was not retained by a cation exchanger. The corn lipid factor had a retention volume of 24 to 30 ml and 22 to 32 ml on a 2.5 ml DEAE column eluted with 1:1 ethanol:H 0 (v/v) with a 0 to 5% 2 acetic acid gradient. Following methylation 75% of the factor was unretained by the DEAE column (eluted with 1:1 ethanol:HZO, v/v), while the unmethylated corn factor was up to 75% retained by the column following the 1:1 ethanol:H 0 2 wash. Following DEAE chromatography, the lipid factor was chromatographed on C18 HPLC and eluted with ethanol:H20:ace- tic acid (80:19.75:0.25, v/v/v) in a retention volume of 4.5 m1. Since the corn factor was lipid soluble and anionic, the chromatographic properties of fatty acids were examined on HPLC using conditions identical to those used for chroma- tography of the corn factor. Stearic, oleic, linoleic and linolenic acids had retention volumes of 6.8, 6.0, and 4.3, and 4.2 ml respectively. Methyl stearate, and methyl palmi- tate had retention volumes of 8.4, and 6.9 ml respectively. 104 As the degree of unsaturation increased for the fatty acids, the retention volumes decreased. The lipid factor had a retention volume similar to that of linoleic and linolenic acids on C18 HPLC (fig. 1). Fatty acids were added to the enzyme assay to determine if they would replace the heat-stable lipid-soluble corn factor in the enhancement of IAA oxidation. Palmitic, stearic, oleic, and linolelaidic acids were inactive in the stimulation of IAA oxidation activity while linoleic, lino- lenic and arachidonic acids, and trilinolenin were stimula- tory (Fig 2,3). Linoleic acid increased enzymatic activity maximally at 3 umol'ml-1 with a 4.1 fold increase in OxIAA formation, while linoleic acid increased enzymatic activity 1 maximally at 0.75 umol'ml- with a 3.1 fold increase. Trilinolenin and arachidonic acid were active in the assay increasing OxIAA formation by 3.45 fold at 1.75 umol'ml'1 and 3.7 fold at 3.0 umol'ml'1, respectively. The trilino- lenin stimulation of enzymatic activity likely results from the lipase catalyzed release of free linolenic acid. Both Triton X100 prepared enzyme, and enzyme prepared without Triton X100 were inhibited by butylated hydroxy- toluene (BHT), cyanide, and mercaptoethanol (table 1). Soybean lipoxygenase I was tested as a model system in the oxidation of IAA. Soybean lipoxygenase plus linoleic or linolenic acids oxidized IAA to a compound with the reten- tion time of OxIAA on C18 HPLC. Soybean lipoxygenase was Fig. 1 Enzyme activity (%increase) 105 50 IinoIonic acid stearic acid r--~ F--n X 40. 30* X 20- 10- " 0 . x . . x—x—x-q—x——1 I r K O 1 2 :3 4» 5 fi- 7 l3 9 10 volume effluent (ml) C18 HPLC of the corn lipid factor, linolenic acid, and stearic acid. The samples were dissolved in ethanol:hexane 1:1 (v/v) and eluted from HPLC with ethanol:H20:acetic acid 80:19.75:O.25 (v/v/v). 106 Fig. 2 The effect of increasing concentrations of linolenic, linoleic, and oleic acids on the enzymatic oxidation of IAA. 107 Fl-E o —°E: me a u p o Linux o «m m. \R .12.. . 6 .0 cm. . m. u. m .. Bun 20.2.: o 33 so... D 0.. N a: 290 9.9.0.2.: W t . 108 Fig 3. The effect of increasing concentrations of trilinolenin (1,2,3 tri-[(cis,cis,cis)-9,12,15 octadecatrienoyl]-rac-glycerol), and arachidonic acid on the enzymatic oxidation of IAA to OxIAA. 109 m_. 1le ./ / 1.90 3:02.395 .}. 5:30:53 0 FI_E._°E-.—. m N N e ,.u19101d Bung-wind (D co m .3“. 110 sensitive to BHT, cyanide, and mercaptoethanol as was the corn enzyme preparation in the oxidation of IAA to OxIAA (table 1). The maximum specific activity of the lipoxy- 1 genase oxidation of IAA was with 1.75 umol'ml' linolenic 1‘ug protein-1). acid (40 pmol OxIAA‘h' Gardner (3)(4) reported that corn germ has hydro- peroxide isomerase and lipoxygenase activities which could be separated from one another by a 0 to 42% and 42 to 53% ammonium sulfate fractionation, respectively. Corn lipox- ygenase, and IAA oxidation activities were nearly equal in each ammonium sulfate fraction, using Gardner's method to concentrate lipoxygenase from 4-day-old endosperm enzyme preparations (0 to 42%, 42 to 53%, and 53 to 80% ammonium sulfate fractionation) (fig. 4). When the experiment was repeated with 0 to 30%, 30 to 42%, 42 to 60%, and 60 to 80% ammonium sulfate fractions, 86% of the lipoxygenase was in the 60 to 80% fraction, and 88% of the hydroperoxide isomer- ase was in the 30 to 42% fraction. However, IAA oxidation to OxIAA was present in all fractions with 40% in the 30 to 42% ammonium sulfate fraction, and 32% in the 42 to 60% ammonium sulfate fraction (fig. 5). Thus, OxIAA formation activity did not co-fractionate with lipoxygenase activity. The IAA oxidase and lipoxygenase activities were not sepa- rated by chromatography on DEAE Sephacryl when eluted with 0.01M phosphate buffer following concentration and desalting by Amicon filtration (data not shown). 111 Table 1. Mercaptoethanol, cyanide, and BHT inhibition of corn enzyme-catalyzed oxidation of IAA, and the soybean lipoxygenase-catalyzed oxidation of IAA. x Source of Enzyme Treatment Endosperm Endosperm Soybean ' (Triton X100) Lipoxygenase mercaptoethanol 1 1 0 cyanide nd 4 5 BHT 200 uM 0 218 nd BHT 20 uM 86 nd 87 a Enzymatic activity is expressed as % of control, nd=not determined, and a enzyme activity was measured in endosperm enzyme preparation plus boiled Triton enzyme preparation 1:1 (v/v). 112 .5 a a: to g C 19 8: X > O X < O ‘t .5} :fifififififififififif c: .— _l E532335333533232;E;E;E;:;:;:;:g:g:;:g:g:; ? C0 0 ‘d “’ E :3 (D B E g, .2 -I: X o E I:I;I;I;Z:.;.;.:.:.;.;.;.;.;.;.;.;.;.;.;.;.;.;.:.;.;.:.:.;.;.;. 5 9; ,g I O I O O O 60— ~ ‘ Runnav 12ml % Fig 4. Ammonium sulfate fractionation (0 to 42%, 42 to 53%, and 53 to 80%) of lipoxygenase and IAA oxidation activities from 4-day-old endosperm enzyme preparations. The totals for IAA oxidation1and lipoxygenase activities were: 1,247 pmol'h' , and 773 AU'min , respectively. 113 IAA oxidation Lipoxygenase U Lipid peroxide isomerase >. :2 .2 ‘6 '1 '5 ‘6 p. 39 _ _:;;:;;:‘._ 42'60 % Ammonium sulfate Fig 5. Ammonium sulfate fractionation (0 to 30%, 30 to 42%, 42 to 60%, and 60 to 80%) of lipoxygenase, hydroperoxide isomerase, and IAA oxidation activity. The totals for enzymatic activities in the ammonium sulfate fractions for IAA oxidation to OxIAA, hydroperoxide isomerase, and lipoxygenase _1 were: 9,619 pmol’h , 108 AU'min , 760 AU'min , respectively. 114 DISCUSSION IAA oxidation to OxIAA was enhanced by a lipid-soluble, heat-stable factor from corn. The corn lipid factor had chromatographic properties similar to that of an unsaturated fatty acid on ion exchange chromatography, and C18 HPLC. The unsaturated fatty acids linoleic, linolenic, and arachi- donic acids replaced the corn lipid factor in stimulating IAA oxidation, although only at high concentrations ranging from nmol to umol'ml'1. The high concentration of fatty acid necessary to oxidize IAA in the in vitro assay suggests that the fatty acids are being metabolized by a competing reaction, or that a fatty acid-like compound with a lower Km than linoleic or linolenic acids is involved in the oxida- tion of IAA to OxIAA. A role for lipoxygenase in IAA oxidation was supported by the specificity in the stimulation of IAA oxidation for a cis,cis,1,4-pentadiene containing fatty acid (2). Oxidation of IAA by soybean lipoxygenase and corn enzyme was inhibited by cyanide, mercaptoethanol, and BHT. The cyanide inhibition indicates an iron group involvement in both reac- tions; the sulfhydryl inhibition may be due to a competitive oxidation of sulfhydryl reagent by lipoxygenase generated free radicals (2) or inhibition due to the reduction state 115. of the enzyme. BHT inhibition of lipoxygenase is thought to occur by reaction of BHT with free or bound free radicals formed in the lipoxygenase reaction (2). IAA oxidation did not correlate with either lipoxygenase or hydroperoxide isomerase when fractionated with ammonium sulfate. Further chromatographic separation of lipoxygenase from the IAA oxidase is required to unequiv- ocally determine whether lipoxygenase is involved in OxIAA formation. Lipoxygenase is the first enzyme in the pathway synthesizing the plant growth inhibitor jasmonic acid (17). In the jasmonic acid pathway, linolenic acid is oxidized to hydroperoxy—linolenic acid, and subsequently cyclized, and oxidized by B-oxidation to jasmonic acid. The knowledge that fatty acids regulate the levels of a growth inhibitor and a plant growth regulator might be important to under- standing how plant growth is controlled. Lipoxygenase is known to co-oxidize carotenes, chloro- phyll, sulfhydryl groups, and fatty acids (2). In a lipox- ygenase co-oxidation reaction, oxidation of IAA would likely be non-specific. Lipoxygenase oxidizes 1-amino- cyclopropane-1-carboxylic acid (ACC) to ethylene by a free radical mechanism (7). The in vivo importance of this reaction is questionable since cell-free systems oxidize 1- amino-2-ethylcyclopropane-1-carboxylic acid, a chiral analog of the natural precursor of ethylene, without the stereo- specificity of intact plant tissues (16). Whether lipox- ygenase oxidizes IAA to OxIAA will be more difficult to 116 determine by stereochemistry studies since IAA has no chiral carbon, and OxIAA has a chemically labile chiral carbon which rapidly exchanges a hydrogen (Paul Lewer, personal communication). A novel "peroxygenase" enzymatic activity was observed in pea microsomes which oxidized indole to 3-hydroxyindole (indoxyl) with linoleic acid hydroperoxide (0.06 mM kM) as co-substrate (5). Oxygen 18 linoleic acid hydroperoxide labeling studies resulted in 63% [180] labeling of the 3- hydroxyindole. An in vivo function for lipoxygenase in the generation of lipid peroxides for the "peroxygenase" cata- lyzed oxidation of lipid soluble molecules was suggested by the authors. The in vivo substrate for the reaction is unknown although indole, analine, phenol, and 1-naphthol were oxidized by the enzyme. If a peroxygenase reaction were involved in IAA oxidation, [180]fatty acid hydro- peroxides would label OxIAA with [180], while a lipoxygenase co-oxidation reaction would not. Recently, the dicot Vicia faba has been shown to oxidize IAA with carboxyl retention to dioxindole-3- acetylaspartic acid, and 3-0-B-glucosyl—dioxindole-3- acetylaspartic acid (15). Interestingly, indole-3- acetylaspartic acid and not free dioxindole-3-acetic acid is an intermediate in the catabolic pathway. The in vitro mechanism of the dioxindole-3-acetic acid pathway has not been elucidated. 117 The first enzyme in the catabolic pathway catalyzing the oxidation of IAA to OxIAA, 7-OH-OxIAA, and 7-OH-OxIAA-glc requires a fatty acid-like factor for maximum activity. Soybean lipoxygenase's oxidation of IAA, and the inhibition of the corn enzyme's oxidation of IAA by BHT suggest a free radical mechanism for IAA oxidation to OxIAA. Further chromatographic characterization of the corn enzyme is required to clarify lipoxygenase's involvement in the oxidation of IAA to OxIAA. 118 LITERATURE CITED Axelrod B, TM Cheesbrough, S Laakso 1981 Lipoxygenase from soybeans. In: Methods in Enzymology. Academic Press, New York, pp 441-451 Gailliard T 1978 Lipolytic and Lipoxygenase enzymes in plants and their action in wounded tissues. G Kahl, ed, Biochemistry of Wounded Plant Tissues. Walter de Gruyter and Co, New York, pp 155-201 Gardner HW 1970 Sequential enzymes of linoleic acid oxidation in corn germ: lipoxygenase and linoleate hydroperoxide isomerase. J of Lipid Research 11:311-321 Gardner HW, D Weisleder 1970 Lipoxygenase from Zea mays: 9-D-hydroperoxy-trans-10,cis-12- ___ octadecadienoic acid from linoleic acid. Lipids 5:678-683 Ishimaru A, I Yamazaki 1977 Hydroperoxide-dependent hydroxylation involving "H 20 -reducible hemoprotein" in microsomes 20? pea seeds. JBC 252: 6118- 6124 Lewer P, RS Bandurski 1986 Occurrence of 7-hydroxy- oxindole-3-acetic acid in seedlings of Zea mays. Plant Physiol 80(S)95 Lynch DV, S Sridhara, JE Thompson 1985 Lipoxygenase- generated hydroperoxides account for the non- physiological features of ethylene formation from 1-aminocyclopropane-1-carboxylic acid by membranes of carnations. Planta 164:121-125 Nonhebel HM, RS Bandurski 1984 Oxidation of indole- 3- acetic acid and oxindole- 3- acetic acid to 2, 3- dihydro- 7- hydroxy- 2- oxo- 1H indole- 3- acetic acid-71- 0- B- D- glucopyranoside in Zea mays seedlings. Plant Physiol 76:979-983 Nonhebel HM, A Crozier, JR Hillman 1983 Analysis of C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse-phase high performance liquid 10. 11. 12. 13. 14. 15. 16. 17. 119 chromatography. Physiol Plant 57:129-134 Nonhebel HM, JR Hillman, A Crozier, MB Wilkins 1985 Metabolism of [ C]indole-3-acetic acid by coleoptiles of Zea mays L. J Exp Bot 36:99-109 Reinecke DM, RS Bandurski 1981 Metabolic conversion of C-indole-3-acetic acid to C-oxindole-3-acetic acid. Biochem Biophys Res Commun 103:429-433 Reinecke DM, RS Bandurski 1983 Oxindole-3-acetic acid, an indole-3-acetic acid catabolite in Zea mays. Plant Physiol 71:211-213 Reinecke DM 1986 In vitro oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme system from Zea mays. Plant Physiol 80(S)118 Reinecke DM, RS Bandurski 1987 Oxidation of Indole-3- acetic acid to Oxindole-3-acetic acid by an enzyme preparation from Zea mays. Doctoral Thesis Experimental section II Tsurumi S, S Wada 1986 Dioxindole-3-acetic acid conjugates formation from indole-3-acetylaspartic acid in Vicia seedlings. Plant Cell Physiol 27:1513-1522 Venus MA 1984 Cell-free ethylene-forming systems lack stereochemical fidelity. Planta 162:85-88 Vick BA, DC Zimmerman 1983 The biosynthesis of jasmonic acid: a physiological role for plant lipoxygenase. Biochem Biophys Res Commun 111:470-477 APPENDICES APPENDIX A Enzymatic activity as a function of germination time Enzymatic activity in endosperm tissue was examined during 0 to 6 days of germination. Seedlings were germi- nated on paper towels in darkness, and enzyme prepared from tissue homogenized in 0.05 M phosphate buffer, 5% PVP (w/w tissue), and 0.04% Triton X100 (v/v) as previously described in the Experimental section. A buffer to tissue ratio of 1:2 (v/tissue) was used in these experiments (1.3 to 1.4 ml:g fresh weight, or 2.5 ml:g fresh weight for day 0 kernels). Enzymatic activity per endosperm was very low in dry kernels (day 0) and day 1 imbibed kernels (fig. 1). Enzyma- tic activity increased to a maximum at day 3 and remained constant through day 6. The specific activity increased to a maximum at day 4 and declined through day 6 (fig. 2). Synthesis of the unknown oxidation product followed a simi- lar profile as OxIAA synthesis, although the maximum rate was shifted a day earlier than the OxIAA synthesis maximum. These data suggest that two separate enzymes are involved in the oxidation of IAA in endosperm tissue. 120 121 Figure 1. Total enzymatic activity for IAA oxidation to OxIAA and to an unknown IAA catabolite as a function of days of germination. IAA oxidation products were assayed as previously described following a 13 h incubation period. n1... 1 o L 5505...: m _ <35 9.255.505 “.0 got .v _ m N O N v ,-wledsopua-,-u-|owd D .H meaawm 123 Figure 2. Specific activity of Triton X100 prepared enzyme as a function of days of germination. 124 o m |I_T L cougar—tom 3 gap v m N P L _ c3052.: (<30 .N cleave . P on N ,_ugeio.1d 6w-,_q-|ould I [ V 125 The day 3 to 5 maximum for enzymatic synthesis of OxIAA could be due to the increased synthesis of the enzyme, or the increased synthesis of a regulatory component, as for example, the lipid factor. Alternatively, the enzyme prepa- ration might be more stable in older seedlings; however, this seems unlikely since proteolytic enzymes increase during germination in storage tissues (1). Root growth commenced on day 1 and shoot growth commenced on day 1 to day 2 for the paper towel grown seedlings. IAA oxidation activity remained high during the time of rapid shoot and root growth, day 2 through day 6. The level of free IAA in corn endosperm remained essentially constant during 4 days of germination (3). The level of free IAA decreased by an estimated 1.8 fold from 1 1 136 1 25 ug'g fresh weight' to 75 i 7 ug'g fresh weight- (n=6) from day 0 to day 2. The decrease in free IAA at day 2 correlates with the initial increase of IAA oxidation activity observed at day 2 in this study. The decrease in IAA concentration might also be explained by a decreased hydrolysis of ester IAA to free IAA in endosperm tissue, or an increased transport rate of IAA from endosperm tissue into vegetative tissue. Enzyme preparations for other experiments described in this thesis were prepared routinely from 4 day germinated corn seedlings due to the high specific activity for IAA oxidation at day 4. APPENDIX B Tritium exchange experiment Corn seedlings germinated on deuterium oxide (2H 0) 2 incorporate deuterium into de novo synthesized molecules. Normal germination occurs with 20 to 30% 2H 0, but at a 2 reduced shoot and root growth rate (9). Two studies, Pengelly et a1 (9), and Scharer and Rimbaut (11), demon- strated that there was incorporation of 2H into IAA during corn seedling germination in 2H20. Pengelly observed that there was 1 to 3 2H incorporated into the indole nucleus, with 19% and 23% of the IAA molecules labeled with heavy isotope for root and shoot, respectively, following 4 days of germination. However, Rimbaut observed only 1 deuterium incorporated into the IAA nucleus per molecule. The discrepancy between the two papers has not been resolved. Deuterium feeding studies give only qualitative data on the occurrence of a de novo pathway, since the effect of mass discrimination on the pathway must be determined to quati- tate heavy isotope feeding studies. Ester conjugated IAA has been shown to be transported from endosperm to vegetative tissues (2) (3) where it may serve as a source of free IAA, the active form of the 126 127 hormone. Vegetative tissue has hydrolases which can 2 hydrolyze ester IAA (4) to free IAA. The H O germination 2 studies give evidence that de novo synthesis of the indole ring occurs during germination, and that there are two sources of IAA for growth in vegetative tissues: de novo synthesis, and hydrolysis of endosperm ester IAA. A question which needs to be addressed is whether the 2H20 studies could have overestimated de novo synthesis due to an enzymatic exchange of 1H for 2 H without indole ring synthesis, as in the phosphate exchange reaction with ATPase (5). The oxidation of IAA to OxIAA could be such a reaction because a hydrogen is added to the 3 position and is removed from the 2 position of the indole ring (Fig. 5 Literature review). To examine whether an enzymatic exchange reaction is involved, a corn enzyme preparation oxidizing IAA to OxIAA was incubated in the presence of 3H 0. 2 Tritiated water study. In the assay 20 mCi'ml-1 of 3H 20 was diluted 1:1 with the enzyme preparation to a -1 specific radioactivity of 180 moi'mole"1 (10 mCi'ml x 1 1 1 O'mole' = 180 mCi'mole-1, 18 g'mole'1 m1 g H20 2 is used in the equation since 3H in the sample is less than X 18 g H 1% of the 1H). If 100% of the IAA (10_u M) added to the 1H, 40,000 DPM of 3H would be incor- 1 assay exchanged 3H for 1 1 porated (180 mCi'mole' x 2.2x109 DPM'mCi- x 10"7 mole'ml' 4 1 IAA = 4.0x10 DPM 'ml- ). If however, the exchange rate only occurred at the rate of IAA oxidation to OxIAA, 1320 DPM of 3H would be incorporated into the indole nucleus 128 1 x 3.3% turnover = 1.3x103 DPM'ml-1). In (40,000 DPM'ml- either case, an efficient method of separating the high 3H O 2 background from the 3H incorporated into OxIAA and IAA is required. The recovery yields would have to be sufficiently high to observe the lower estimate of incorporation of 1320 DPM. The method would also need to employ a base exchange step since IAA and OxIAA have labile protons in the acetic acid side chain which can non-enzymatically incorporate 3H (6). Enzyme assay. Five hundred ul of Triton X100 endosperm enzyme preparation was diluted with 0.5 m1 3H 0 plus 0.1 mM 2 IAA, or with 0.5 ml H 0 plus 0.1 mM IAA and 1.8 pM [1-1uCJ- 2 IAA. The assays along with boiled enzyme controls were 0 C in a waterbath incubated in 10 ml screw cap vials at 30 with shaking. After a uh incubation period, the 3H20 assays were chilled on ice and acidified to pH 3.0 with concen- trated acetic acid. One mg each of OxIAA and IAA was added to the assay mixture for recovery estimation during purifi- cation. The HZO-[11uCJ-IAA assays were terminated by the addition of 2 ml of acetone. Isolation of labeled IAA and OxIAA. The HZO-[114CJ-IAA assays were chromatographed on a Varian 5000 HPLC, C18 column 0.H6x25 cm, and eluted with ethanol:H2 20:79:1 (v/v/v) at 1 ml'min'1. OxIAA synthesis was 3.3 i 1 O:acetic acid 0.4% of the IAA added or 3.3 nmol'ml- (3.3% turnover X 10'7 1 IAA - 3.3 nmoi'mi'1 mole'ml' OxIAA). 129 The 3H20 assays were passed through a C18 Sep Pak (Waters) to separate unreacted 3H 0 from the IAA and OxIAA. 2 The Sep Pak was prewashed with 0 ml of methanol and 4 ml of H20:acetic acid 99:1 (v/v). Care was taken to prevent the Sep Pak from drying out during the prewash and the sample application. The samples were injected onto the Sep Pak and eluted n times with 2 m1 H20:acetic acid 99:1 (v/v) (solvent 1), and 3 times with 2 m1 ethanol:H20 8:2 (v/v) (solvent 2). Preliminary experiments showed that less than 0.002% of the 3H20 remained on the column following solvent 1 elution, and 90 to 97% of the IAA was recovered following solvent 2 elution. The eluant from solvent system 2 was evaporated to the aqueous phase and taken up in ethanol:H20:acetic acid 20:79:1 (v/v/v) for chromatography on HPLC as described previously for the HZO-[11uCJ-IAA assay. Five hundred ul fractions of eluant were collected and measured for radio- activity by scintillation counting. Absorbance at 280 nm for IAA and 254 nm for OxIAA was measured on a Gilford spectrophotometer. Fractions absorbing at 280 nm or 254 nm were pooled and further purified separately as follows. IAA and OxIAA have an acetic acid side chain which exchanges protons under basic conditions, while the ring system protons are stable (6). The samples plus 10% KOH (w/v) were added to a Teflon pressure bomb. Following the addition of 5 mg of sodium dithionite and flushing with argon to reduce oxidation losses, the pressure bomb was 130 O C for 1 h. The samples were cooled and heated to 120 partitioned with ether. The samples were then acidified to pH 2.7 and partitioned with ether. The acid ether fractions containing either IAA or OxIAA were taken to dryness, resus- pended in H20:acetic acid 99:1 (v/v), and passed through a C18 Sep Pak to remove 3H 0 as previously described. The IAA 2 and OxIAA fractions were pooled and chromatographed on C18 HPLC as previously described. One ml fractions were collected and radioactivity was measured in fractions absorbing at 280 or 254 nm. The recovery of IAA from the assays was 47 to 65%, and the radioactivity recovery in the IAA fraction was only 9 to 11% showing that base catalyzed 3H exchange had occurred. The recovery of radioactivity in the OxIAA fraction was 7 to 9%, and the recovery of OxIAA was not determined since OxIAA ring expands to 1,2,3,“ tetrahydroquinolone-M-carboxylic acid under basic conditions. The 1,2,3,4 tetrahydro- quinolone-N-carboxylic acid was further purified on HPLC by 10 um Partisil 10 (silica, 0.46x25 cm) eluted with a gradient of ethanol:ethyl acetate:hexane 0:“5:55 (v/v/v) to 10:35:55 (v/v/v) over 10 min at 2 ml'min-1. Fractions eluting at retention times of 13 to 15 min were collected and radioactivity measured by scintillation counting. IAA recovered from the base exchange-C18 HPLC step was methyl- ated by ethereal diazomethane (12) and chromatographed on 1 C18 HPLC eluted with ethanol:HZO 1:1 (v/v) at 1 ml'min- . 131 Fractions eluting at 6.5 to 7.5 min retention times were collected and radioactivity measured. Results. There was no radioactivity above background in either the methyl-IAA following C18 HPLC, or the 1,2,3,” tetrahydroquinolone-u-carboxylic acid (OxIAA) following silica HPLC. The methyl-IAA fractions had no peak of radio- activity corresponding to the 280 nm IAA peak, and the CPM were only 40 to 76 CPM above background. The 1,2,3,4 tetrahydroquinolone-u-carboxylic acid had no peak of radio- activity associated with 254 nm absorbance, and the CPM were 75 to 220 CPM above a blank control. The boiled control in the OxIAA experiment had more radioactivity associated with it than the the enzyme assay while both had similar recov- eries. The final recovery of IAA for the purification method was 22 to 23%. Although these experiments can not totally rule out enzyme catalyzed exchange, IAA exchange must be very small under these experimental conditions since the difference between the enzyme assay and the boiled control is only 130 DPM, while 100% exchange would have resulted in u0,000 DPM incorporation (4H8 DPM boiled control X 0.23-1 recovery - M00 DPM enzyme assay X 0.22-1 recovery : 130 DPM). Throughout the purification method a full UV spectrum of the 280 and 254 nm absorbing fractions were routinely recorded on a Cary 15 spectrophotometer to confirm the presence of IAA and OxIAA in the fractions collected. The recovery of radioactivity in the IAA and 132 OxIAA fractions is shown for the enzyme assays and the boiled controls in (Table 1). Subsequently, it has been shown by NMR that the 3 position of the oxindole nucleus is labile and readily exchanges 2H at room temperature when 7-0H-0xIAA was dissolved in 2 H20 (P. Lewer personal communication). This explains why OxIAA had higher 3H incorporation than IAA at every step of the purification method. The facile exchange of 2H on the oxindole ring makes meaningful information concerning OxIAA synthesis difficult to obtain by heavy isotope experiments. The protons on the indole ring are stable, and such experiments may clarify the role of de novo synthesis for IAA. To examine whether 3H may be discriminated against in the enzyme catalyzed reaction, the experiment could be 2 H20 using the same incubation method and purification procedure. performed by the incubation of enzyme preparations on Heavy isotope incorporation could be monitored by GC-MS. Trace amounts of radiolabeled IAA could be added to the reaction mixture to facilitate peak hunting. Alternatively, seedlings could be grown on 3H 0 and examined for heavy 2 2 isotope incorporation into IAA as was observed for H 0. 2 The isolation and characterization of the enzyme system which synthesizes IAA would permit an exact evaluation of mass discrimination in indole synthesis during growth on isotopically labeled water. The present studies suggest 133 that enzyme catalyzed proton exchange does not significantly interfere with 2H20 estimation of de novo synthesis of IAA. Table 1. 134 The 3H radioactivity associated with IAA and OxIAA at various stages of purification following incubation of corn endosperm enzyme preparation The IAA recoveries for the enzyme Essay and the boiled control were within The OxIAA recoveries for the enzyme assay and the boiled control were within with 1% through step 3. 3% through step a. H 0 and IAA. IAA OxIAA boiled enzyme boiled enzyme control assay control assay Total DPM 1. C18 HPLC 164,700 158,000 ”12,000 397.000 (after assay) 2. C18 HPLC 13,900 16,500 13,300 10,700 (base exchanged) 3. C18 HPLC 400 448 --- --- (methylated IAA) M. silica HPLC --- --- 2845 1786 APPENDIX C IAA and OxIAA effect on enzymatic activity The kinetics of IAA oxidation to OxIAA were examined by incubating corn enzyme preparation with increasing amounts of IAA or OxIAA. Preliminary kinetic studies were initiated with the crude enzyme preparation to determine the optimum concentration of IAA for the assay, and to determine if OxIAA accumulation during the assay would result in feedback inhibition of the IAA oxidation reaction. Assay. The enzyme assay included 0.9 ml corn enzyme prepared with or without Triton X100, 1.7 to 1.9 nmol of [1- 1"01111111, and 0.1 ml of appropriately diluted IAA or OxIAA. The enzymatic activity was assayed following a uh incubation period, and OxIAA synthesized was determined as previously described. 6 to 10"3 Results. IAA oxidation was active over a 10' M concentration range (Table 2). The rate of increase of enzymatic activity was slower with increasing amounts of IAA; however, enzymatic activity was not substrate saturable under these conditions. Enzyme prepared with and without Triton X100 showed similar enzymatic activity profiles. As the [1uC]IAA was diluted with increasing amounts of 135 136 Table 2. The effect of increasing IAA concentration on the oxidation of IAA. Catabolites synthesized are expressed as nmol/ml of assay medium. IAA added nmol OxIAA nmol unknown flit synthesized catabolite 1.9 x 10"6 0.15 0.03 1.0 x 10‘5 0.7a 0.11 1.0 x 10’” 3.85 0.25 1.0 x 10"3 19.1 0.90 unlabeled IAA, the per cent of [1uCJIAA oxidized decreased (7.62% of the undiluted [1”CJIAA was oxidized to [1”010xIAA, while 1.91% of the [14C]IAA diluted to 10"3 M with unlabeled IAA was oxidized to [1”010xIAA). Undiluted [1-1MCJIAA was routinely used in OxIAA quantitation assays to obtain the highest sensitivity in the assay. Tritiated IAA has a higher specific radioactivity than [1uCJIAA and would increase the sensitivity of the assay. Tritiated IAA was not used due to the interference of [3H] enzymatic and non- enzymatically decarboxylation contaminants. The addition of unlabeled OxIAA at the beginning of the incubation period only slightly inhibited the oxidation of IAA (Table 3). OxIAA added at 1000 fold higher concen- tration than the enzymatically synthesized OxIAA resulted in only 16% inhibition of IAA oxidation. The synthesis of the unidentified IAA catabolite was essentially unaffected by increasing amounts of unlabeled OxIAA supporting the 137 [3H]OxIAA data that OxIAA was not the metabolite's precursor. Table 3. The effect of increasing OxIAA concentration on IAA oxidation. The enzyme preparation did not include Triton X100 nmol pmole OxIAA pmole unknown OxIAA added synthesized synthesized 0 16.7 16.7 0.83 16.2 15.7 3.35 15.0 16.2 16.6 14.0 16.4 APPENDIX D ENZYMATIC ACTIVITIES OF SEEDLING TISSUES IAA oxidation to OxIAA was examined in several corn tissues including the coleoptile, the apical and basal meso- cotyl, and the endosperm; the cortex and stele mesocotyl tissues, and endosperm and scutellum tissues. Morpho- logically a corn shoot includes a modified leaf sheath, the coleoptile, which surrounds the juvenile corn leaves. The corn mesocotyl is a shoot internode located between the coleoptile and the corn kernel. Running longitudinally through the mesocotyl is the stele, a transport tissue made up of xylem and phloem, and surrounding the stele is the cortex and epidermis of the mesocotyl. In dark grown seedlings most of the shoot growth occurs in the mesocotyl. The corn kernel is composed of three major tissues: the starchy endosperm--the bulk of the kernel, the scutellum-- the absorptive tissue which contains the majority of the kernel's storage lipids, and the aleurone layer which surrounds the endosperm and synthesizes hydrolytic enzymes. Assays. For the corn shoot-endosperm experiments, 3 g fresh weight of tissue was homogenized with 3.5 ml of 0.05 M phosphate buffer plus 0.4% Triton X100 (v/v) and 5% PVP 138 139 (w/w). Homogenization with an Omni mixer resulted in foaming of vegetative preparations and was replaced with grinding by mortar and pestle in the vegetative experi- ments. The apical and basal mesocotyl preparations included the upper and lower 1 cm sections of the mesocotyl (3.76:0.5 cm n=25), and the coleoptile preparations included the coleoptile and the enclosed primary leaves (1.09:0.19 cm). Mesocotyl cortex was separated from the stele as previously described (10). Endosperm preparations included the endo- sperm and aleurone layer. A buffer to fresh weight ratio of 5:0.59 and 6:4 were employed for the stele-cortex experi- ments and for the scutellum-endosperm tissue experiments, respectively. Corn shoot-endosperm enzyme preparations were diluted to 2 mg protein'ml'1 for enzyme assays. The extracts were centrifuged and dialyzed as previously described. Enzyme assays were run for 4 h at 300 C, and OxIAA synthesis was measured by an HPLC-scintillation counting method (Experimental Section 1, Materials and Methods). Results. The stele and cortex enzymatic activities were proportional to protein concentration from 0.5 to 1.7 1 and 0.1 to 0.95 mg’ml'1, respectively. The mg'ml' scutellum preparation's activity was proportional to protein concentration up to 5 mg protein'ml'1 and endosperm plus scutellum up to 2.4 mg protein’ml'1, the maximum concen- trations assayed. The apical mesocotyl, the most actively growing shoot tissue (10) had the highest specific activity 140 for IAA oxidation being 9.1 and 1.4 times higher than coleoptile and basal mesocotyl tissues (Table 4). The total enzyme activity per tissue was 2.5 fold higher in the apical 1 cm than in the basal 1 cm of the mesocotyl. Corn endosperm (plus scutellum) tissue had higher specific and total activities than vegetative tissues. Scutellum tissue had 4.2 times higher total activity per g fresh weight than endosperm (minus scutellum) (Table 5b); scutellum tissue was the best source of high specific activity enzyme preparation for oxidation of IAA to OxIAA, 1'mg protein-1. The function for the with up to 80 pmol'h' high in vivo turnover of IAA in corn endosperm and scutellum remains unknown. While the specific activities in the stele and cortex were similar, the total activity per tissue in the cortex was 7 times higher (Table 5a), supporting Nonhebel's [‘“CJIAA feeding studies (7) which showed that IAA was actively metabolized in the growing cortical tissues of roots, and slowly metabolized in stele transport tissues. Interestingly, shoot mesocotyl tissue decarboxylated 85 to 90% of the IAA during a 4h incubation period while Nonhebel (8) observed no substantial loss of radioactivity from feeding [1-1uCJIAA to corn shoot segments. The in vitro assay unmasks unphysiologically high rates of IAA decarboxylation. Conclusions. These preliminary tissue experiments give evidence for higher rates of IAA oxidation in actively Table 4. Tissue apical mesocotyl basal mesocotyl endosperm 141 Enzymatic activities for coleoptile, apical and basal mesocotyl, and endgsperm tissues. Recovery refers to recovery18f [ C] following incubation of enzyme with [1- CJIAA. Specific Total Total Recovery Activity Activity Activity pmol'h'1; pmol'h'1’ pmol'h-l' per cent mg prot' g fwt' tissue- 0.92 12.4 0.31 92.7 8.37 67.5 1.82 14 5.88 21.3 0.72 9.7 9.02 39.2 12.5 89 142 growing vegetative tissues: the apical mesocotyl and mesocotyl cortex. The high rates of IAA oxidation in endosperm and scutellum tissues may suggest a role for IAA metabolism in remobilization of storage materials during germination in corn. 143 Table 5. Enzymatic activities determined for (a) cortex and stele of corn mesocotyl, and (b) corn endosperm and scutellar tissues. nd = not determined. Specific Total Total Recovery Activity Activity Activity Tissue pmol'h-li pmol'h'1‘ pmol‘h::° per cent mg prot g Fwt' tissue (a) stele 9.0 77.4 0.77 10.1 cortex 10.8 65.4 5.55 14.8 (b) scutellum nd 38.3 12.6 100 plus endosperm endosperm nd 14.5 3.6 100 minus scutellum scutellum nd 61.0 4.6 29 10. LITERATURE CITED Ching, TM 1972 Metabolism of germinating seeds. 13: Seed Biology. Volume II/Germination, metabolism, and Pathology. ed TT Kozlowski, Academic Press, New York Chisnel JR, RS Bandurski 1983 Translocation of 5-3H-IAA and 5- H-IAA-myo-inositol from vegetative tissue of Zea mays. Plant Physiol 72:827 Epstein E, JD Cohen, RS Bandurski 1980 Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol 65:415-421 Hall PJ, RS Bandurski 1986 [3HJIndole-3—acetyl-myo- inositol hydrolysis by extracts of Zea mays L. vegetative tissue. Plant Physiol 80:374-377 Ingle J, L Beevers, RH Hageman 1964 Metabolic changes associated with the germination of corn I. Changes in weight and metabolites and their redistribution in the embryo axis, scutellum, and endosperm. Plant Physiol 39:735-740 Lehninger, AL 1975 In: Biochemistry. The molecular basis of cell structure and function. Worth Publishers, New York Magnus V, RS Bandurski, A Schulze 1980 Synthesis of 4,5,6,7 and 2,4,5,6,7 deuterium-labeled indole-3- acetic acid for use in mass spectrometric assays. Plant Physiol 66:775-781 Nonhebel HM, JR Hillman, A Crozier, MB Wilkins 1985 Metabolism of [ C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots. Planta 164:105-108 Nonhebel HM, JR Hillman, A Crozier, MB Wilkins 1985 Metabolism of [ C]indole-3-acetic acid by coleoptiles of Zea mays L. J Exp Bot 36:99-109 Pengelly WL, RS Bandurski 1983 Analysis of indole-3- acetic acid metabolism in Zea mays using deuterium oxide as a tracer. Plant Physiol 73:445-449 144 145 11. Pengelly WL, PJ Hall, A Schulze, and RS Bandurski 1982 Distribution of free and ester indole-3-acetic acid in the cortex and stele of the Zea mays mesocotyl. Plant Physiol 1304-1307 12. Scharer S, JM Rimbaut 1983 Biosynthese de l'AIA dans les plantules entieres et les racines excisees de Zea mays. Experimental work for the Plant Physiology Certificate, University de Lausanne 13. Schlenk H, JL Gellerman 1960 Esterification of fatty acids with diazomethane on a small scale. Anal Chem: 32:1412-1414 BIBLIOGRAPHY BIBLIOGRAPHY Abbot MT, 8 Udenfriend 1974 Alpha-ketoglutarate-coupled dioxygenase. In: Molecular mechanisms of oxygen activation. ed 0 Hayaishi pp 167-214 Academic Press, New York Allen W 1969 The incorporation of oxygen-18 into oxindole acetic acid by cells of Hygrophorus conicus. MS thesis. University of Missouri-Rolla Axelrod B, TM Cheesbrough, S Laakso 1981 Lipoxygenase from soybeans. In: Methods in Enzymology. Academic Press, New York, pp 441-451 Bandurski RS, A Schulze 1977 Concentration of indole-3- acetic acid and its derivitives in plants. Plant Physiol 60:211-213 Bandurski RS, A Schulze, D Reinecke 1985 An attempt to localize and identify the gravity sensing mechanism of plants. The Physiologist 28:8-111-112 Bandurski RS, HM Nonhebel 1984 Auxins. In: Advanced Plant Physiology. ed M Wilkins, Pitman Press, London, pp 1-20 BeMiller JN, W Colilla 1972 Mechanism of corn indole-3- acetic acid oxidase in vitro. Phytochem 11:3393- 3402 Ching, TM 1972 Metabolism of germinating seeds. In: Seed Biology. Volume II/Germination, metabolism, and Pathology. ed TT Kozlowski, Academic Press, New York Chisnell JR RS Bandurski 1983 Translocation of 5-3H-IAA and 5- H-IAA-myo-inositol from vegetative tissue of Zea mays. Plant Physiol 72:827 Cohen JD, RS Bandurski 1978 The bound auxins: Protection of indole-3-acetic acid from peroxidase-catalyzed oxidation. Planta 139:203-208 Cohen JD, BG Baldi, JP Slovin 1986 13C -[Benzene ring]- indole-3-acetic acid. A new inte nal standard for 146 147 quantitative mass spectral analysis of indole-3- acetic acid in plants. Plant Physiol 80:14-19 Cohen JD, A Schulze 1981 Double standard isotope dilution assay. I. Quantitative assay of indole-3- acetic acid. Anal Biochem 112:249-257 Davies PJ 1972 The fate of exogenously applied indoleacetic acid in light grown stems. Physiol. Plant. 27:262-270 Engvild KC, H Egsgaard, E Larson 1980 Determination of 4-chloro-3-indole-3-acetic acid methyl ester in Lathyrus, Vicia and Pisum by gas chromatography- mass spectrometry. Physiol Plant 48:499-503 Epstein E, JD Cohen 1981 Microscale preparation of pentafluorobenzyl esters: electron capture gas chromatographic detection of indole-3-acetic acid from plants. J Chromatogr 209:413-420 Epstein E, JD Cohen, RS Bandurski 1980 Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol 65:415-421 Gailliard T 1978 Lipolytic and Lipoxygenase enzymes in plants and their action in wounded tissues. G Kahl, ed, In: Biochemistry of Wounded Plant Tissues. Walter de Gruyter and Co, New York, pp 155-201 Galston AW, J Bonner, RS Baker 1953 Flavoprotein and peroxidase as components of the indoleacetic acid oxidase system of peas. Arch Biochem Biophys 42:456-470 Galston AW, HR Chen 1965 Auxin activity of isatin and oxindole—3-acetic acid. Plant Physiol 40:699-705 Galston AW, WS Hillman 1961 The degradation of auxin. In: Hanbuch der Pflanzen Physiologie. ed W Ruhland, Springer-Verlag, Berlin, pp 647-670 Gardner HW, 1970 Sequential enzymes of linoleic acid oxidation in corn germ: lipoxygenase and linoleate hydroperoxide isomerase. J of Lipid Research 11:311-321 Gardner HW, D Weisleder 1970 Lipoxygenase from Zea mays: 9-D-hydroperoxy-trans-10,cis-12-octadecadienoic acid from linoleic acid. Lipids 5:678-683 Grambow HJ, B Langenbeck-Schwich 1983 The relationship between oxidase activity, peroxidase activity, 148 H 0 , and phenolic compounds in the degradation of ifidgle-3-acetic acid in vitro. Planta 157:131-137 Greenwood MS, JR Hillman, S Shaw, and MB Wilkins 1973 Localization and identification of auxin in roots of Zea mays. Planta 109:369-374 Hall PL, RS Bandurski 1978 Movement of indole-3-acetic acid and tryptophan-derived indole-3-acetic acid from the endosperm to the shoot of Zea mays L. Plant Physiol 61:425-429 Hall PJ, RS Bandurski 1986 [BHJIndole-3-acetyl-myo- inositol hydrolysis by extracts of Zea mays L. vegetative tissue. Plant Physiol 80:374-377 Hedden P, JE Graebe 1982 Cofactor requirements for the soluble oxidases in the metabolism of the C20- gibberellins. J Plant Growth Regul 1:105-116 Henderson JHN, CS Patel 1972 0xindole-3-acetic acid: physical properties and lack of influence on growth. Physiol Plant 27:441-442 Hinman RL, CP Bauman 1964 Reactions of N- bromosuccinimide and indoles. A simple synthesis of 3-bromooxindoles. J Org Chem 29:1206-1215 Hinman RL, J Lang 1965 Peroxidase catalyzed oxidation of indole-3-acetic acid. Biochem 4:144- 157 Ingle J, L Beevers, RH Hageman 1964 Metabolic changes associated with the germination of corn I. Changes in weight and metabolites and their redistribution in the embryo axis, scutellum, and endosperm. Plant Physiol 39:735-740 Ishimaru A, I Yamazaki 1977 Hydroperoxide-dependent hydroxylation involving "H20 -reducible hemoprotein" in microsomes o? pea seeds. JBC 252:6118-6124 Julian PL, HC Printy, R Ketcham, R Doone 1953 Studies in the indole series XIV oxindole-3-acetic acid. J Am Chem Soc 75:5305-5309 Kaufman S, DB Fisher 1974 Pterin-requiring aromatic amino acid hydroxylases. In: Molecular mechanisms of oxygen activation. 0 Hayaishi (ed) pp 285-349 Academic Press, New York 149 Kenten RH 1955 The oxidation of indolyl-3-acetic acid by waxpod bean root sap and peroxidase systems. Biochem. J. 59:110 Kinashi H, Y Suzuki, S Takeuchi, A Kawarada 1976 Possible metabolic intermediates from IAA to B-acid in rice bran. Agric Biol Chem 40:2465-2470 Klambt HD 1959 Die 2 Hydroxy-Indol-3-essigsaure, ein pflanzliches Indolderivat. Naturwiss 46:649 Klambt HD 1964 2-hydroxyindole-3-acetic acid and similar compounds in seeds and other plant parts. In: Regulateurs Naturels de la Croissance Vegetale, pp235-239, ed JP Nitsch, CNRS, Paris Kogl FA, J Haagen-Smit, H Erxleben 1934 Uber ein neues auxin (heteroauxin) aus harn. Zeit Physiol Chem 228:104-112 Kokkinakis DM, JL Brooks 1979 Hydrogen peroxide; mediated oxidation of indole-3-acetic acid by tomato peroxidase and molecular oxygen. Plant Physiol. 64:220-223 Langenbeck-Schwich B, HJ Grambow 1984 Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments. Physiol. Plant. 61:125-129 Lehninger AL 1977 Biochemistry. The molecular basis of cell structure and function. Worth Puplishers Inc, New York Lewer P, RS Bandurski 1984 Occurrence of 7-hydroxy- oxindole-3-acetic acid in seedlings of Zea mays. Plant Physiol 80(S)95 Lynch DV, S Sridhara, JE Thompson 1985 Lipoxygenase- generated hydroperoxides account for the non- physiological features of ethylene formation from 1-aminocyclopropane-1-carboxylic acid by membranes of carnations. Planta 164:121-125 Magnus V, RS Bandurski, A Schulze 1980 Synthesis of 4,5,6,7 and 2,4,5,6,7 deuterium-labeled indole-3- acetic acid for use in mass spectrometric assays. Plant Physiol 66:775-781 Magnus V, S Iskric, S Kveder 1971 Indole-3-methanol — A metabolite of indole-3-acetic acid in pea seedlings. Planta 97:116-125 Mehta JM 1968 A study of succinic dehydrogenase, indole acetic acid oxidase, nitrate reductase and 150 cytochrome c reductase in Hygrophorus conicus. PhD. University of Missouri-Rolla Micha czuk L, JR Chisnell 1982 Enzymatic synghesis of 5- H- indole- 3- acetyl-myo- inositol from 5 tryptophan. J Labelled Compd Radiopharm 19:121-128 Nakono M, S Kobayashi, K Sugioka 1982 Peroxidase catalyzed oxidation of indole-3-acetic acid. In: Oxygenases and oxygen metabolism. pp 245-254 Academic Press Nickell LG 1983 In: Plant Growth Regulating Chemicals. vol 1 and vol 2 CRC Press, Boca Raton, Florida Nitsch JP, C Nitsch 1956 Studies on the growth of coleoptile and first internode sections. A new sensitive, straight-growth test for auxins. Plant Physiol 31:94-111 Nonhebel HM 1982 The metabolism of indole-3-acetic acid in seedlings of Zea mays L. PhD thesis. University of Glasgow Nonhebel HM 1986 Measurement of the rates of oxindole-3- acetic acid turnover, and indole-3-acetic acid oxidation in Zea mays seedlings J Exp Bot 37:1691- 1697 Nonhebel HM, RS Bandurski 1984 Oxidation of indole- 3- acetic acid and oxindole- 3- acetic acid to 2, 3- dihydro- 7- hydroxy- 2- oxo-1H indole- 3- acetic acid-71- 0- B- D- glucopyranoside in Zea mays seedlings. Plant Physiol 76:979-983 Nonhebel HM, A Crozier, JR Hillman 1983 Analysis of C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse- phase high performance liquid chromatography. Physiol Plant 57:129-134 Nonhebel HM, JR Hillman, A Crozier, MB Wilkins 1985 Metabolism of [ C]indole-3-acetic acid by coleoptiles of Zea mays L. J Exp Bot 36:99-109 Nonhebel HM, JR Hillman, A Crozier, MB Wilkins 1985 Metabolism of [ C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots. Planta 164:105-108 Patterson B, 1965 Studies on the conversion of indole acetic acid to oxindole acetic acid by Hygrophorus conicus. MS thesis. University of Missouri-Rolla 151 Pengelly WL, RS Bandurski 1983 Analysis of indole-3- acetic acid metabolism in Zea mays using deuterium oxide as a tracer. Plant Physiol 73:445—449 Pengelly WL, PJ Hall, A Schulze, and RS Bandurski 1982 Distribution of free and ester indole-3-acetic acid in the cortex and stele of the Zea mays mesocotyl. Plant Physiol 1304-1307 Pless T, M Bottger, P Hedden, J Graebe 1984 Occurrence of 4-Cl-indoleacetic acid in broad beans and correlation of its levels with seed development. Plant Physiol 74:320-323 Rayle DL, RE Cleland 1970 Enhancement of wall loosening and elongation by acid solutions. Plant Physiol 46:250-253 Reinecke DM 1986 In vitro oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme system from Zea mays. Plant Physiol 80(S)118 Reinecke DM, RS Bandurski 1981 Metabolic conversion of C-indole-3-acetic acid to C-oxindole-3-acetic acid. Biochem Biophys Res Commun 103:429-433 Reinecke DM, RS Bandurski 1982 Quantitation of oxindol- 3-yl-acetic acid in Zea mays. Plant Physiol 69:3- 307 Reinecke DM, RS Bandurski 1987 Oxidation of indole-3- acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays. Doctoral thesis Experimental section II Reinecke DM, RS Bandurski 1983 Oxindole-3-acetic acid, an indole-3-acetic acid catabolite in Zea mays. Plant Physiol 71:211-213 Reinecke DM, RS Bandurski 1984 Oxidation of indole-3- acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays seedlings. Plant Physiol 75(8):108 Reinecke DM, RS Bandurski 1985 Further characterization of the enzymatic oxidation of indole-3-acetic acid to oxindole-3-acetic acid. Plant Physiol 77(S)3 Reynolds, JD, TD Kimbrough, B Weekley 1983 Evidence for enzymatic 5-hydroxylation of indole-3-acetic acid in vitro by extracts of Sedum morganianum. Z. Pflanzenphysiol. Bd. 112 465-470 152 Rittenberg D, GL Foster 1940 A new procedure for quantitative analysis by isotope dilution with application to the determination of amino acids and fatty acids. J Biol Chem 133:737-744 Sabater F, J Sanchez-Bravo, M Acosta 1983 Effects of enzyme/substrate ratio and of cofactors on the oxidation of indole-3-acetic acid catalyzed by peroxidase. Revista Espanola de Fisiologia 39:169- 174 Sandberg G 1984 Biosynthesis and metabolism of indole-3- ethanol and indole-3-acetic acid by Pinus Sylvestris L. needles. Planta 161:398-403 Sandberg G, E Jensen, A Crozier 1984 Analyses of indole- 3-carboxylic acid in Pinus sylvestris needles. Phytochem. 23:99-102 Scharer S, JM Ribant 1983 Biosynthese de l'AIA dans les plantules entieres et les racines excisees de Zea mays. Experimental work for the Plant Physiology Certificate, Universite de Lausanne Schlenk H, JL Gellerman 1960 Esterification of fatty acids with diazomethane on a small scale. Anal Chem 32:1412-1414 Schneider EA, F Wightman 1978 Auxins. In: Phytohormones and Related Compounds: A Comprehensive Treatise. DS Letham, PB, Goodwin, TJV Higgins (eds), 1:29-105. Amsterdam, Elsevier/North-Holland Schuytema EC, MP Hargie, I Merits, JR Schenck, DJ Siehr, MS Smith, EL Varner 1966 Isolation, characterization, and growth of basidiomycetes. Biotech & Bioeng 8:275-286 Sembdner G, D Gross, H-W Liebisch, G Schneider 1981 Biosynthesis and metabolism of plant hormones. In: Hormonal Regulation of Development. I. Molecular Aspects of Plant Hormones (Encyclopedia of Plant Physiology Ser: Vol. 9), J MacMillan (ed),pp. 281- 444. Berlin: Springer-Verlag Siehr DJ 1961 The formation of oxindole-3-acetic acid from indoles by a basidiomycete. J Am Chem Soc 83:2401-2402 Sundberg, B, R Sandberg, E Jensen 1985 Identification of indole-3-methanol in etiolated seedlings of Scots Pine (Pinus sylvestris L.). Plant Physiol 77:952- 955 153 Sundberg B, G Sandberg, and E Jensen 1985 Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris). Physiol Plant 64:438-444 Suzuki Y, H Kinashi, S Takeuchi, A Kawarada 1977 (+)-5- Hydroxy-dioxindole-3-acetic acid, a synergist from rice bran of auxin-induced ethylene production in plant tissue. Phytochem 16:635-637 Theologis A, TV Huynh, RW Davis 1985 Rapid induction of specific mRNAs by auxin in pea epicotyl tissue. J Mol Biol 183:53-68 Thimann, KV 1934 Studies on the growth hormone of plants. VI. The distribution of the growth substance. J Gen Phys 18:23-34 Torti G, L Lombardi, LA Manzocchi, F Salamini 1984 Indole-3-acetic acid content in viable defective endosperm mutants of maize. Maydica 29:335-343 Tsurumi S, S Wada 1980 Metabolism of indole-3-acetic acid and natural occurrence of dioxindole-3-acetic acid derivatives in Vicia roots. Plant and Cell Physiol 21:1515-1525 Tsurumi S, S Wada 1985 Identification of 3-(O-B- glucosyl)-2-indolone-3-acetylaspartic acid as a new indole-3-acetic acid metabolite in Vicia seedlings. Plant Physiol 79:667-671 Tsurumi S, S Wada 1986 Identification of 3-hydroxy-2- indolone-3-acetylaspartic acid as a new indole-3- acetic acid metabolite in Vicia roots. Plant Cell Physiol 27:559-562 Tsurumi S, S Wada 1986 Dioxindole-3-acetic acid conjugates formation from indole-3-acetylaspartic acid in Vicia seedlings. Plant Cell Physiol 27:1513-1522 Venis, MA 1984 Cell-free ethylene-forming systems lack stereochemical fidelity. Planta 162:85-88 Vick BA, DC Zimmerman 1983 The biosynthesis of jasmonic acid: a physiological role for plant lipoxygenase. Biochem Biophys Res Commun 111:470-477 Vijayaraghavan SJ, WL Pengelly 1986 Bound auxin metabolism in cultured Crown-gall tissues of tobacco. Plant Physiol 80:315-1321 154 Waldrum JD, E Davies 1981 Subcellular localization of IAA oxidase in peas. Plant Physiol 68:1303-1307 Weber F, W Grosch 1976 Co-oxydation of a carotenoid by the enzyme lipoxygenase: influence on the formation of linoleic acid hydroperoxides. Z Lebensm Unters- Forsch 161:223-230 Weis JS 1966 Effects of oxindoles on the growth of tobacco tissue cultures. Nature 211:1216-1217 Went FW, KV Thimann 1978 Phytohormones. Reprinted 1978 Allanheld,Osmun/Universe Books, New York Wightman F, DL Lighty 1982 Indentification of phenylacetic acid as a natural auxin in the shoots of higher plants. Physiol Plant 55:17-24