§§ WWII”WIHIHHWIHHHIIHHIWWIHW‘HI ITHS_ THESIS _L A ~ ‘— LIE RA HY Michig an State 1 University \ ‘1 This is to certify that the thesis entitled THE CATABOLISM OF INDOLE-3-ACETIC ACID 1 IN ZEA MAYS ENDOSPERM ",1, ““pTE'SEfited by DENNIS M. REINECKE has been accepted towards fulfillment of the requirements for , QMSQMWL Major professor Date_AUfillSI_5._19.8.L_ 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to move charge fran circulation records THE CATABOLISM OF INDOLE-3-ACETIC ACID IN ZEA MAYS ENDOSPERM BY Dennis M. Reinecke A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1981 CH; 4%/ ABSTRACT THE CATABOLISM OF INDOLE-3-ACETIC ACID IN ZEA MAYS ENDOSPERM BY Dennis M. Reinecke l4C]-oxindole-3-acetic acid was identified as a cata- [l- bolic product of [1-14C1-indole-3—acetic acid metabolism in Egg gays seedlings. IAA catabolic products were purified by the following chromatographic sequence: DEAE-cellulose, DEAR-Sephadex, LH-20 Lip0philic Sephadex, and HPLC. The putative oxindole-B-acetic acid cochromatographed with authentic oxindole-3-acetic acid in all chromatographic sys— tems tested, and gas chromatography-mass spectrometry con- firmed the identity of the catabolic product as oxindole-B— acetic acid. Oxindole-B-acetic acid accounted for, at least, 26% of the expected carboxyl retaining IAA catabolism of endosperm tissue without allowance for further metabolism. The amount of oxindole-3-acetic acid was determined to be 73 ug/lOOO kernels or 356 ug/kg dry wt.' This is the first identification of oxindole-3-acetic acid as a major cata- bolic product of IAA, and the first quantitation of oxindole- 3-acetic acid in plant tissue. ACKNOWLEDGMENTS I would like to acknowledge Dr. Robert Bandurski for the opportunity to work on this project and for his encouragement. The assistance of my committee members Dr. Mathew Zabik and Dr. Derek Lamport is also appreciated. I would like to thank Prudy Hall for her help with the mass spectrometry, and Jerry Cohen for his assistance and interest. I would also like to acknowledge Dr. Richard Barr who sparked my interest in plant physiology as an undergraduate at Shippensburg State College. Finally, I want to thank my family and friends, especially Carol and Denny, for their support through the writing of this thesis. ii TABLE OF CONTENTS Page LIST OF TABLES 0 O O O O O O 0 O 0 O O O O V LIST OF FIGURES . . . . . . . . . . . . . vi LIST OF ABBREVIATIONS . . . . . . . . . . . Vil INTRODUCTION . . . . . . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . 10 Plant Material . . . . . . . . . . . . . 10 Chromatographic Material . . . . . . . . . . lO Spectrophotometric Determinations . . . . . . . 11 Chemicals . . . . . . . . . . . . . . ll Radiological Material . . . . . . . . . . 11 Determination of Specific Activity . . . . . . 11 Synthesis of [13C] -Carboxyl Labeled IAA . . . . . 12 [1-13CJ- IAA Labeling Experiments . . . . . . l4 Isolation of OxIAA from Zea ma 5 Endosperm Tissue . 18 Quantitation of Oxindole- 3- acet1c Acid . . . . . 18 EXPERIMNTAL O O I I O C O O O O O O O O 25 Metabolic Conversion of l4C-indole-B-acetic Acid to 4C-oxindole-B—acetic Acid . . . . . . . . 25 RESULTS o o o o o o o o o o e o o o o o 31 Identification of Oxindole—3-acetic Acid . . . . 31 Turnover of Labeled IAA . . . . . 34 Quantitation of Oxindole- 3- acetic Acid in Zea mays Endosperm Tissue . . . . . . . . . . . . 37 DISCUSSION . . . . . . . . . . . . . . . 39 Oxindole— 3- acetic Acid as a Major Route of IAA Catabolism . . . . . . . . . . . 40 Indole- 3- acetic Acid Turnover . . . . . 4l Biological Activity of Oxindole- 3-acetic Acid . . . 42 Identification of OxIAA . . . . . . . . . . 46 iii Page SUMMARY . . . . . . . . . . . . . . . . 50 BIBLIOGRAPHY . . . . . . . . . . . . . . 52 iv LI ST OF TABLES Table Page 1. Chromatographic similarities between authentic and putative OxIAA . . . . . . . . . . 32 2. Major GC-MS fragments and the percentage relative abundances for authentic and putative OxIAA. . 35 LIST OF FIGURES Figure Page INTRODUCTION 1. Oxidative pathways of indole-3—acetic acid catabolism . . . . . . . . . . . . 6 2. The catabolic turnover of indole-3-acetic acid in Zea endosperm tissue . . . . . . . . 9 MATERIALS AND METHODS 3. The synthesis of [13C]-carboxy1 labeled indole— 3-acetic acid from gramine . . . . . . . 15 4. The 70 eV mass spectrum of the methyl ester of [13c1-carboxy1 labeled indole—3—acetic acid . 16 5. The extraction procedure for the isolation of carboxyl retaining indole—3-acetic acid catabolites . . . . . . . . . . . . l9 6. The isolation and identification scheme for carboxyl retaining indole-3-acetic acid catabolites . . . . . . . . . . . . 20 EXPERIMENTAL 7. The 70 eV mass spectra of the pentafluorobenzyl ester of authentic oxindole-B-acetic acid (a), and that of the putative oxindole-B-acetic acid isolated from Zea endosperm (b). . . . 28 DISCUSSION 8. The keto and enol forms of zeanic acid and B- acid . . . . . . . . . . . . . . 44 9. The acid and base rearrangement products of OxIAA and dioxindole—3-acetic acid . . . . 45 vi BSTFA DEAE GLC GC-MS HPLC IAA m/z OxIAA PFB TLC TMS UV LIST OF ABBREVIATIONS Bis-(trimethylsilyl)trifluoroacetamide diethylaminoethyl gas-liquid chromatography gas chromatography-mass spectrometry high pressure liquid chromatography indole-3-acetic acid mass to charge ratio oxindole-B-acetic acid pentafluorobenzyl thin—layer chromatography trimethylsilyl ultraviolet vii INTRODUCTION The development of the growth hormone concept in plants can be traced back over one hundred years to the pioneering work of Ciesielski (8) studying the geotropic response in roots, and Darwin (11) studying the phototropic response in coleoptiles. These workers observed that removal of the tip tissue resulted in loss of trOpic sensitivity, suggest- ing that the tip was a source of a transitory growth stimulus which regulated growth and tropic responses. Subsequently, indole-3-acetic acid was isolated and identified as a plant growth hormone (41, 24). To understand how the hormone regulates growth, it is essential to understand how metabolism regulates the level of hormone. In detipped A3333 coleoptiles, Bonner and Thimann (6) correlated growth with the inactivation of hor- mone. More recent studies have indicated that the level of hormone may be regulated by catabolism (23), compartmentation (4), conjugation (l), and synthesis (57). In the endosperm of 4 day old dark-grown seedlings, the plant system studied in this work, indole—3-acetic acid (IAA) is not being con- jugated, but is rapidly turning over through an unidentified catabolic route. The following discourse will be limited to the examination of how IAA is catabolized in various plant systems, and to the elucidation of the IAA catabolic pathway inéssms- Kisser et al. (38) and Thimann (65) were the first to observe that crushed leaves and water extracts of leaves, respectively, inactivated the growth promoting substance. The inactivation of the hormone was shown to be enzymatic and to require oxygen by Larsen (43). Galston et a1. (21) demonstrated that the IAA oxidizing system of pea extracts, first reported by Tang and Bonner (14), had a peroxidase and a flavin component. Galston's group also showed that crystalline horseradish peroxidase had a similar IAA oxidiz- ing activity. There are many papers reporting the inactivation of IAA in plant systems, and these are discussed in detail in the review articles of Galston and Hillman (23), Schneider and Wightman (55), Sembdner et al. (57), and Hare (27). These studies measured IAA catabolism colorimetrically, most often using the Ehrlich (15), and Salkowski reagents (52); manometrically, measuring CO2 evolution or 02 uptake; with bioassay; or radiologically. With the exception of the radio- logical studies, none of these methods was sufficiently sen- sitive to measure the amount of IAA "used up" by the tissue during growth. Care must be exercised in the comparison of the cata- bolism of exogenously applied IAA in whole tissues, crude homogenates, and enzyme preparations to the catabolism of endogenous levels of IAA in vivo. Also, one must be careful to exclude nonenzymatic destruction of IAA by UV radiation, strong acids, and oxidizing agents (23, 32, 33). Deverall (13) reported the decarboxylation of IAA in a buffered solution, pH 4.5-4.7, approaching 20% decarboxylation in 1 hour, dependent on the washing treatment of the glassware. Bacterial contamination (2), and the concentration of IAA (32) may change the profile and extent of IAA catabolism also. Several studies have followed radioactive l4CO2 evo- lution of IAA labeled in the l, 2, and ring positions. Andreae et a1. (2) observed 19% decarboxylation of [1-14C]- labeled IAA with pea root tips over 24 hours with 10"4 M IAA. Andreae reported that 17% of the IAA was conjugated to IAA aspartate, and 6% was degraded without carboxyl loss. Only negligible decarboxylation of [2-14C] and [7a-14C1-label was observed in this system, although up to quantitative release 14 of CO was observed from the 7a labeled IAA when the 2 incubation mixture was contaminated with microorganisms. Davies (12) estimated that the amount of decarboxylation of IAA in kidney bean and pea segments was 34% and 29%, respec- tively, over 6 hours of incubation. Troxler and Hamilton (66) with geranium callus cultures, and Strydom and Hartman (61) with plum stem cuttings observed 32% and 31% of the [2-14C]-1abeled IAA as respired l4CO2 after 7 and 1 day incubations, respectively. However, Hamilton et a1. (26) working with a crown-gall tissue culture of Parthenocissus tricuspidata observed only 1% loss of [2-14C1-1abe1, and 53% loss of [1-14C1-label over a 48 hour incubation period. Epstein and Lavee (18) observed that the age of the culture influences the amount of decarboxylation in apple tissue culture. Three month old nongrowing apple callus cultures quickly decarboxylated 90% of the [1-14C1-labeled IAA during 4 hours, while young, growing callus cultures decarboxylated only 20% of the [1—14CJ-labeled IAA during 4 hours. Fang and Butts (19) found that corn seedlings had a light require- ment for the decarboxylation of [1-14C]-labeled IAA applied to primary leaves, with negligible degradation of IAA in the dark. The leaves of pea and bean decarboxylated IAA in both the light and the dark, but more slowly in the dark. Excised corn shoots only decarboxylated 12-19% of labeled IAA over 6 hours in the light, and 8-13% in the dark; while corn root tips decarboxylated 82% in the light, and 85% in the dark (20). Pea tissues were more active in degrading IAA, with pea shoots decarboxylating 80-90% of the labeled IAA in the light, and 54-80% in the dark; and pea roots quantitatively decarboxylating labeled IAA in the light or the dark. These experiments demonstrated that there can be qualitative and quantitative differences in the destruction of IAA in different plant species, and there may be quali- tative and quantitative differences in the destruction of IAA in different tissues of the same plant. Many studies of IAA oxidation have indicated that IAA was oxidatively decarboxylated, but it was the work of Hinman and Lang (32) with an ER zitrg horseradish peroxidase system that elucidated the major products as oxindole-3— carbinol (hydroxymethyloxindole), and methyleneoxindole (Figure 1a) by means of UV spectroscopy, and chemical ana- lOgue studies. Hydroxymethyloxindole and methyleneoxindole were subsequently reported as products of IAA oxidation in pea (68) and corn extracts (5). Other reported decarboxyla- tion products of IAA catabolism include: indolealdehyde (50), methyloxindole (68), indole-B—methanol (45), indole-3- carboxylic acid (45), 3—acetoxyindole, and oxindole-indole polymers (62). Whether these compounds are of physiological significance is uncertain, since many of the studies were performed in_vitro with high IAA concentrations. Kinashi and coworkers (37) have isolated several com- pounds from rice bran which are possible IAA catabolites that retain the carboxyl group, including: the methyl esters of oxindole-3-acetic acid and dioxindole-3-acetic acid, the 5-hydroxy analogues of the aforementioned compounds, and 5- hydroxy-oxindole—3-acetic acid (Figure lb). Klambt (39) has presented colorimetric evidence for the occurrence of oxindole-3-acetic acid (OxIAA) in seeds and seedlings of Zea mays, in germinating seeds of Brassica rapa, and in devel- oping seeds of Ribes rubrum. Klambt also reported the isolation of the glucoside of OxIAA (40) from feeding [l-l4Cl- IAA to several plant species, but his use of ammoniacal solvents in the chromatographic separations makes the isolation of a labile glucose ester impossible (34). Siehr (58) has .cflom oaumomumnmaovcflxoflo can pflom oaumomumlmaowsflxo Op ddH mo :oflumoflxo .ma0©:axoocoahnumelm can maovsflxoahnuofihxouchsum ou ¢¢H mo coaumaaxonumomc m>wumvflxo .M .Emflaonmumo oequMImlmHoccfl mo mmm3numm o>HDMUHxOII.H musmflm 2.80.88.85.26... use u..o8-m.o.856 I I on _ / 9T2 _ / :ooouxo _ \ rooomzo _ \ :o I .1 NC... a / m_ou=_xooc§£§-m Secretariat?» Iooomzu _ _ .1 N8 - \ d shown that the basidiomycete Hygrophorus conicus can metab- olize tryptamine and IAA to OxIAA. The quinolinic acids, B-acid (2,6-dihydroxycinchonic acid) in rice bran (37), and zeanic acid (2,8-dihydroxycinchonic acid) in corn (47), are other possible catabolites from IAA which retain the carboxyl group. However, B-acid and possibly zeanic acid are arti- facts of isolation since dioxindole-B-acetic acid, and oxindole-3-acetic acid rearrange in the presence of base or acid to similar compounds (35, 67, 39, 63). Tsurumi and Wada (67) have reported dioxindole-B-acetic acid derivatives in Vicia faba from IAA transport studies. KOpcewicz et a1. (42) observed several apparent oxi- dation products of IAA from a 24 hour incubation of mature l4C]-IAA. The products corn kernels in a solution of [1- did not yield IAA or indoleacetamide upon ammonolysis, but further chemical identification was not undertaken. Epstein et a1. (17) collected 14CO2 evolved from [1-14C1—IAA applied to the cut endosperm of 4 day old dark-grown corn seedlings. Only 12 pmol/h/endosperm of IAA was decarboxylated. Isotope dilution experiments demonstrated that IAA remained rela- tively constant over 4 days of growth at 308 pmol/endosperm. Turnover studies showed that the specific activity of labeled IAA was diluted by an apparent first order rate over 8 hours. From these data on dilution of specific activity over time, the first order rate constant, k, of the reaction was determined to be 0.22/h and the turnover time was cal— culated to equal 3.2 h. Since there were 308 pmol/endosperm, the turnover rate was calculated to be 96 pmol/h/endosperm. Of this rapidly turning over IAA pool, only 12 pmol/h was decarboxylated, so that 83 pmol/h of IAA catabolism was through an unidentified carboxyl retaining catabolic route (Figure 2). In the following experiments the carboxyl retaining IAA catabolites are isolated and identified, as the first step towards understanding the function of the rapid turnover of IAA in endosperm tissue and its role in growth. I2 pmol-h" 83 pmol~h:\? / I I / 96 pmol-h" / I * | CHZCOOH ‘ . l ‘coz< N Figure 2.--The catabolic turnover of indole-3-acetic acid in Zea endosperm tissue. MATERIALS AND METHODS Plant Material Seeds of Zea mays cv. Stowell's Evergreen Sweet corn were purchased from Vaughan Jacklin Co., Ovid, Mi., and W. Atlee Burpee Co., Clinton, Iowa. Chromatographic Material Thin-layer chromatography was on Silica Gel 60 TL plates without fluorescent indicator (E. Merk, Darmstadt, Germany). DEAE-cellulose coarse mesh, DEAE-Sephadex A-25-120, and lipophilic Sephadex LH-20—100, were purchased from Sigma. A Whatman Partisil 10 ODS 25 x 0.46 cm C18 column was employed for high pressure liquid chromatography (HPLC). A Hewlett—Packard model 402 was used for preliminary gas-liquid chromatography (GLC) on*a 1.2 m x 2 mm ID 3% OV-l on Gas Chrom Q (100/120) column (Applied Science Laboratories, State College, Pa.). Subsequent work was performed on a Varian Series 2700 gas chromatograph with a 1.8 m x 2 mm ID OV-l on Gas Chrom Q (100/120) column, or 3% OV-l7 on Gas Chrom Q (60/80) column (Applied Science Laboratories, State College, Pa.). A Hewlett-Packard 5985 gas chromatograph- mass spectrometer (GC-MS) using a 1.8 m x 2 mm ID 3% SP2250 10 11 on Supelcoport (80/100) column (Supelco, Inc., Bellefonte, Pa.) was employed for GC—MS analysis. Spectr0photometric Determinations A Gilford 240 Spectrophotometer, and a Cary 15 spectrophotometer were used for spectrophotometric analysis. Chemicals IAA, Sigma Chemical Co., St. Louis, Missouri; gramine, Diazald (N-methyl-N-nitroso-p-toluenesulfonamide), 2-(2- ethoxy)-ethanol,m-bromopentafluorotoluene, and dimethyl sulfate, Aldrich Chemical Co., Milwaukee, Wisconsin; potas- sium cyanide [13C] 90 atom %, Merk, Sharp, and Dohme, Rahway, NJ; N-ethylpiperidine, Pfaltz and Bauer, Flushing, NY; N-bromosuccinimide, Eastman Organic Chemicals, Rochester, NY; Bis—(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% Trimethylchlorosilane, Regis Chemical Co., Chicago, Il. Radiological Material [1-14CJ-IAA (57 uCi/umol and 58 uCi/umol) was purchased from Amersham, Arlington Heights, 11. Radioactivity was measured on a Packard Tri—Carb liquid scintillation spectro- meter, and on a Beckman LS 7000 liquid scintillation counter. ACS (Amersham) was used as the scintillation cocktail. Determination of Specific Activity Specific activity (uCi/umol) was determined by measuring radioactivity by liquid scintillation counting, and concen- trations by UV absorbance. Concentrations were determined 12 by measuring the absorbance of the solution of interest on a Cary 15 spectrophotometer. The absorbance and the known molar extinction coefficient may be used in the Beer-Lambert law: A = ccl absorbance where: A c = molar extinction coefficient (liters/mole- centimeter) c = concentration (moles/liter) l = pathlength (centimeters) The radioactivity of the solution was measured by liquid scintillation counting and corrected for efficiency by an 4 internal [1 C]-toluene standard, or an external [137Cs] standard. Synthesis of [13C]-Carboxyl Labeled IAA Stowe (60) described a method for the synthesis of high specific activity [14C]-carboxy1 labeled IAA by reacting gramine methosulfate (Sch6pf & Thesing) with K14CN. The product, indoleacetonitrile was then hydrolyzed to yield [1-14C]-labe1ed IAA. By using K13 CN, the reaction was modi- fied for the synthesis of [1-13CJ-1abeled IAA. The reactive tertiary amine gramine methosulfate was synthesized at 1/10 scale using the method of Schdpf and Thesing (56). Gramine was recrystallized from hot acetone and hexane; tetrahydrofuran was redistilled over potassium and under nitrogen; acetic acid was dried over P205 and redistilled, and dimethyl sulfate was redistilled under 13 reduced pressure. For the synthesis, 0.5 mmol acetic acid was mixed with a solution of 14 mmol gramine and 25 m1 of peroxide-free tetrahydrofuran. The mixture was added drOp- wise with stirring to a solution of 0.1 mol of dimethyl sulfate and 0.5 mol of acetic acid in 10 m1 of tetrahydro- furan at 16°C in the dark. After 30 minutes the reaction flask containing a white oil was transferred to a dark room at 5°C. Crystallization occurred only after the oil was washed with anhydrous ethyl ether. The crystals melted initially at 135—137°C, and l4l-l43°C after drying over P205. Stowe reported a melting point of 146-148°C for the product and yields of 87-98%. I obtained 4.0 grams of gramine methosulfate for a 96% yield based on gramine. For the synthesis of labeled IAA, 70 mg of gramine methosulfate plus 15 mg of K13CN were added to a 5 m1 round bottomed flask. Next, 2 ml of 0.2 M K HPO 2 4 nitrogen, and then added to the flask. The mixture was was purged with agitated with a nitrogen stream through a glass capillary while the reaction vessel was heated to 65°C for 5 hours. One pellet of KOH and boiling chips were added and the mix- ture refluxed for 1 hour. The contents were filtered through a sintered glass funnel into a 10 ml beaker, and the flask and filter washed with two 0.5 m1 aliquots of H O. The 2 filtrate was chilled to ice temperature and acidified to pH 3.5 with concentrated phosphoric acid, whereupon thick white crystals formed. The crystals were washed twice with 0.5 m1 of distilled H O, and then recrystallized from 2 14 95% ethanol. Melting occurred at 164-166°C with the reported value for the melting of IAA being l65-166°C. The yield of [13CJ-carboxy1 labeled IAA was 33% based on [13C1-cyanide. Stowe reports a yield of between 20-80% under similar con- ditions. A summary of the synthesis of gramine methosulfate and [13C]-1abe1ed IAA is given in Figure 3. The UV spectrum of the putative IAA had a peak at 282 nm and 220 nm as did authentic IAA. The ratio of the molar extinction coefficients of IAA at 282 nm to 222 nm, 6060/33,200 (3), equals 0.183 which is identical to the ratio of the absorbances of the putative IAA at 282 nm to 220 nm, with 0.268/l.465 equaling 0.183. The putative IAA cochroma- tographed with authentic IAA on TLC in the solvent system chloroform:methanol:water 85:14:1, and the putative IAA was Ehmann positive as was authentic IAA (14). The mass spectrum of the methyl ester of the putative IAA had a molecular ion at 190, and characteristic fragment ions at 130, 103, and 77 while authentic IAA had a molecular ion 189, and character- istic fragment ions at 130, 103, and 77. Figure 4 shows the fragmentation pattern of the methyl ester of the [13C]- labeled IAA. l [l- 3C]-IAA Labeling Experiments Initial feeding experiments were conducted using [1-13C]-labe1ed IAA at a ratio of 1:1 with the endogenous free IAA. The [1-13CJ-labeled IAA plus a small amount of [1-14C]-IAA, as an easily followed tracer, was applied to 15 .msflemum Eoum neon oauoomumloaoncw ooaonma meonumouHUmaa mo mflmonucam m£B|I.m ousmam OU< vzwulxtmédog 332203943002. I Z// \v//, 1. L of _ , xooonhxo _ a : zonwzo L/\ 209 x w»(m._DmQ:w<m on m£BII.e onsmfim c- SN sow so“ 2: at on; 03 01 m2 mm“ a: of mm. mm. a. ...................... .::.::_:E:F::E:._::.::_:_:::_::::L»:J.r: EerE—Eztttflt :55 E 1‘ no. 1 C)m Y b N om. U fr I D [s on. 3 7: K) AllSNBlNI BALLV‘ISH 9t. 17 the cut endosperm of 4 day old dark-grown §g§_may§ seedlings. All manipulations were at 25°C, 80% relative humidity, using a phototropically inactive green safe light (17). After a 4 hour incubation period the corn kernels were excised and homogenized in enough acetone to make the final concentration 70% allowing for the water content of the kernels. The carboxyl retaining IAA catabolites were purified by a sequence of partitionings with l-butanol, and chloroform; and by column chromatography with DEAE-cellulose, DEAE- Sephadex, and LH-20 lipOphilic Sephadex. IAA catabolites which retained the carboxyl group were to be identified by gas chromatography—mass spectrometry. Since [1—13C1- labeled IAA was added to the tissue in a 1:1 ratio to the endOgenous free IAA, carboxyl retaining catabolites would have distinctive double—peaked molecular ions of similar intensity by gas chromatography-mass spectrometry. The method of identifying carboxyl retaining IAA catabolites by the double-peaked mass spectral marker proved unsuccessful owing to the occurrence at 1000 fold greater concentration of the plant phenolic compounds (3). Typi- cally, IAA and IAA carboxyl retaining catabolites lose the carboxyl label at the first fragmentation in GC-MS. The double label would only be detected in a clean sample where the doubled-peaked molecular ion could be clearly distin- guished from contaminating fragments. To circumvent this problem the [1-13C1-IAA experiments were suSpended, and labeling experiments with higher specific activity 18 [1-14C1-IAA were begun. The [1-14C1-experiments were initiated to develop better purification procedures for the isolation of IAA catabolites for GC-MS identification. Isolation of OxIAA from Zea mays Endosperm Tissue [1—14C]-labeled IAA was applied to the cut endosperm of 4 day old dark-grown seedlings. After a 4 hour incu- bation period, the kernels were excised and extracted with 70% acetone at -78°C. Figure 5 gives a summary of the initial extractions and partitionings used to isolate the IAA catabolites from the endosperm tissue. The acetone extraction recovered up to 90% of the initial radioactivity added to the tissue, indicating that decarboxylation of the IAA by the tissue was very low. The recovery yield through the butanol partitioning and the chloroform extraction ranged between 60% and 70%. The crude chloroform extract was further purified with DEAE-cellulose, and DEAE-Sephadex anion exchange chromatog- raphy, LH-20 lip0philic Sephadex chromatography, and C18 reverse phase HPLC as summarized in Figure 6. Two peaks of radioactivity were resolved by LH-20 chromatography. The purified, derivatized samples were analyzed on gas chromatoqraphy-mass spectrometry. Quantitation of Oxindole-3-acetic Acid 1 The previous experiments indentified [1- 4C]-OxIAA as a carboxyl retaining catabolic product of [1-14C1-IAA metab- olism in Zea mays. The next questions to answer were 19 CORN SEEDLINGS + lL‘C-IAA I mam WITH 70: ACEmNE RESIDUE FILTRATE BUTANJL PARTITION H20 PHASE BUTANOL PHASE CHLOROFOR‘) EXTRACT RESIIlJE CHLOROFORVI PHASE Figure 5.--The extraction procedure for the isolation of carboxl retaining indole-3-acetic acid catabolites. l PEAK I DERIVATIZE l‘PLC E-IB 20 IIAECELLULDSE IIRIVATIZE GC-l‘B Figure 6.--The isolation and identification of carboxyl retaining indole-3—acetic catabolites. 21 whether OxIAA is a naturally occurring compound in Zea_endo— sperm tissue, and if so, at what concentration does it occur. An external standard method, and the double standard isotope dilution assay were employed to quantitate the endogenous levels of OxIAA. The external standard method compares the GLC peak area of an external standard of known amount, to the GLC peak area of a plant sample of unknown amount: umol external standard peak area ==umol plant sample peak area external standard plant sample eluting from GLC The GLC peak areas of both compounds are quantitated by taking the average weight of three photocopies of each peak tracing. The amount in umoles of external standard eluting from the GLC effluent is calculated by collecting the radio- activity eluting from the extinguished FID at the standard's known retention time. The radioactivity collected, times the known specific activity for the external standard, equals the amount in umoles of standard eluting from the GLC. With this information, the preceding equation can be solved for the amount in umoles of a plant sample required to give the observed GLC peak re3ponse. The amount of OxIAA initially in the tissue can be calculated from the GLC value, by correcting for the recovery yield for the isolation of OxIAA. The main weakness of the external standard method is its dependence on an accurate determination of recovery 22 yield, which may vary depending on the concentration of OxIAA in the tissue. The double standard isotOpe dilution assay was employed for a more exact determination of levels of OxIAA in endo- sperm tissue (10). This method can quantitate labile com- pounds in small amounts such as plant hormones and their catabolites. The double standard isotOpe dilution assay circumvents the difficulties of the external standard method, accurate measurement of yield recovery and accurate quanti- tative gas chromatography, by employing two internal stan- dards (10). The first internal standard is a radioactively labeled standard of the compound to be quantitated in the tissue. The initial specific activity of the standard is determined, and the specific activity of the standard after its addition and reisolation from the tissue is determined. Any OxIAA present in the tissue will dilute the specific activity of an OxIAA standard added to the tissue. The initial specific activity, the final diluted specific activity, and the amount of standard added to the tissue can be measured to determine the endOgenous levels of OxIAA, as in the following isotope dilution equation (51): C 0 Cf 14 specific activity of C-OxIAA added where: C o C specific activity of l4C-OxIAA recovered f 23 amount of l4C-OxIAA added N II Y = amount of endogenous plant OxIAA To determine the final specific activity of the compound to be quantitated, a second internal standard is added to the purified sample prior to gas-liquid chromatography. In the present study IAA is used as the second internal standard. By measuring the radioactivity, and peak areas (as described in the external standard section), the final diluted specific activity of OxIAA may be calculated (10): collected OxIAA (DPM) IAA peak area Xspecific activity OxIAA peak area coIlected IAA (DPM) of IAA = final specific activity OxIAA The final specific activity of the OxIAA thus determined, the known initial specific activity, Co' and the known amount of OxIAA added, X, can be used to solve the isotOpe dilution equation for the endogenous level of OxIAA: C OxIAA in endosperm tissue = ( —9 -1 ) X Cf Labeled OxIAA, 10 uCi/umol, was synthesized for the isotope dilution assay using the method of Hinman and Bauman (30), and further purified on an LH-20 column and eluted with 50% 2-pr0panol. The radiological purity of the synthetic OxIAA was shown to be at least 95% by HPLC eluted with 20% ethanol plus 1% acetic acid, and 94% by TLC devel- oped with G solvent. 24 Both the external standard method and the double stan- dard isotOpe dilution assay depend on clean GLC peaks free from cochromatographing contaminants. The identity of the peak was further verified by GC-MS in the present study. EXPERIMENTAL METABOLIC CONVERSION OF "C-INDOLE-S-ACITIC ACID TO “C-OXINDOLE-SACE’I‘IC ACID MM.MandRob-ts.w Wtdlotanyandl’lanththelogy MIehlganStateUniveulty Bath-slag,Mlehlgan 48824 Received : SUMMARY: We have identified [l-"Cl-oxindole-J-acetic acid as a catabolic product of [lo"C]-indole-3- acetic acid metabolism in Zea mays seedlings. The isolation. and chemical and mass spectral characterization of oxindole-B-acetic acid from corn kernel tissue is described together with data suggesting oxindole-lacetic acid to be a major catabolic product ot'indole-J-aeetic acid. Studies of the geotropic response of roots by Ciesielski (l) and of the phototropic response of shoots by Darwin and Darwin (2) led to the conclusion that a growth stimulus was transmitted backwards from the tip (cf.3). Removal or damage to the tip resulted in loss (1’ tropic sensitivity indicating that the stimulus must be “used up” when the tip of the plant is removed“). . Subsequent research led to the identification of indole-3-aeetic acid (IAA) (S) as a plant growth hormone. and a pathway for catabolism of IAA was discovered which involved decarboxylation of the IAA. typically as catalyzed by horseradish peroxidase in vitro (6). However. among the few plants studied. Zea mays (7) and. possibly. Pisum (8) destroy only a minor portion of the IAA peroxidatively with carboxyl loss. Several IAA-related compounds that retain the carboxyl have been reported in rice bran. including methyloxindole-3- acetic acid. methyl-dioxindoIe-3-acetic acid. the S-hydroxyl analogs of both the aforementioned compounds. and S-hydrorydioxindolc-Ii—acetic acid (9). In addition. Siehr (10) found that a basidiomycete Hygrophorus conicus metabolizes tryptamine and IAA to oxindole-3-acetic acid (OxIAA). and Klimbt (ll) has presented colorimetric evidence for the occurrence of OxIAA in 3 plant species including Zea seedlings. he present work is the first demonstration of the metabolic production of [l-"CI-OslAA fi'om [l-"Cl-IAA in plants. 25 26 MAN AND METHODS lamb: DEAE-Sephadex A-25. DEAE-cellulose coarse mesh. lipophilic Sephadex urea. and indole-3-acetic acid. Sigma Chemical Co.; a-bromopentafluorotoluene. Diazald (hi-methyloN-nitroso-p-toluenesulfonamide). Aldrich; N-ethylpiperidine. Mat: and Bauer; Silica Gel 60 thin-layer plates without fluorescent dye. E. Merck: 3% OV-l7 on Gas Chrom 0 (60M). Applied Science Laboratories; 3% SP-2250 on Supelcoport (80.400). Supelco. lnc.; [l-"Cl-indole-Ii-acetic acid. 57 mCi/mmol. Amersham: and N-bromosuccinimide. Eastman Organic Chemicals. Incubation and Crude Extract Preparation: Corn kernels. Zea mays cv. Stowell's Evergreen Sweet corn 0”. Atlee Burpee Co.). were germinated for four days in darkness at 25’C and 80% relative humidity using a phototropically inactive green safe light for necessary manipulations (7). About We of the endosperm was cut from the end of the kernel and 5 ul of 50% ethanol containing 25 ng of [l-"C]-indole-3-acetic acid (57 mCr/mmol) was applied. This amounts to 46% of the endogenous free IAA contained in a kernel (7). After a four hour incubation of the 1000 treated seedlings. the kernels were excised and dmpped into sufficient acetone at -78°C to make the final acetone concentration 70% allowing for the water in the kernels. This labeled material was used to develop purification and derivatiza- tion techniques. whereas a large incubation mixture prepared from 5am seedlings to which was added approxi- mately 10% of the labeled extract was used for the final chemical characterization of the unknown compounds. In a control experiment. ll-"CHAA was added to the cut endosperm of seedlings. the excised kernels were immediately dropped into acetone at -78°C. and the hornogenate extracted as described. Only unchanged labeled 1AA could be reisolated from the control unincubated seedlings. The incubated kernels were homogenized for two minutes in a four liter Waring blender and extracted at 4°C for 12 hours. The hornogenate was filtered and the residue reextracted two times with 70% acetone using 12 hour extraction periods. Acetone was removed from the filtrates under reduced pressure. and the aqueous phase partitioned three times with l-butanol. The butanol phase was reduced to a yellow paste under reduced pressure. and the yellow paste extracted overnight in chloroform. After filtration. the yellow CHCl. filtrate was evaporated to about X) ml for further purification using column chromatography. Chm-”Why: The extract was chromatographed on a 2.5 x 1) cm DEAE-cellulose column to remove lipoidal material (12). and yielded a single peak of radioactivity eluted with CHCl./CH,OH/CH,COOH 7023021. The components of this peak could be resolved into two zones by thin-layer chromatography (CHCl.:CH,OH:H,O 85:14:” with R, values of 0.20 and 0.40 with standard IAA at R,=0.42. The DEAE-cellulose peak was pooled and chromatographed on a 2.5 ml DEAE-Sephadex column. washed with 50% ethanol. and eluted with a linear gradient from 50% ethanol to 50% ethanol containing 5% acetic acid. The radioactive peak was pooled and chromatographed on a 2.3 x 20 cm LH-ZO column and eluted with 50% Z-propanol. Two peaks of radioactivity eluted from the Lil-20 column and the R, values of these peaks on TLC using the above solvent system were: peak 1 R,=0.l9. peak ll R,=0.42. and standard MA at R,=0.47. The material in peak 11 from LH-X) chromatography was methylated with diazomethane (13). and the resultant ester reacted with bis-(trimethylsilyl)otrifluoroacetamide at 45’C for 15 minutes to derivatize the imine nitrogen. The pentafluorobenzyl ester of the material in peak I was synthesized using o-bromo- pentafluorotoluene in the presence of N-ethyipiperidine (14) and purified by HPLC on a Cu reverse phase column using 50% ethanol as eluant. The 70 eV mass spectral fragmentation pattern of these derivatives was analyzed with a Hewlett-Packard 5%5 GC-MS using a 1.8m x 2mm ID SP2250 column programmed from 220-280°C. 27 RESULTS W of cub-M add: OxIAA was synthesized by oxidation of [AA to OxIAA with 1 mole equivalent of N-brornosuccinimide [caution-explosion hazard (15)] according to the method of Hinman and Bauman (16) at 1/ 1m scale with addi- tion of 2 Ni of [1-"Cl-1AA. The resultant OxIAA was purified by Lil-20 chromatography with 50% 2-propanol as eluant. and by HPLC using 20% ethanol plus 1% acetic acid as eluant. The following criteria indicated the product to be OxIAA: the UV spectrum ofthe product in 95% ethanol evidenced a peak at 248 nm. and a shoulder at 280 nm; the product was unreactive with Ehmann reagent (17); a green color was produced with Ehrlich reagent afier TLC of the product using 2-propanol:NH.OH:l-1,0 80:15:5 as solvent (12): and the product retained the labeled carboxyl group. The 70 eV mass spectrum of the pentafluorobenzyl ester proved the product to be oxindole-B-acetic acid with a molecular ion at m/e=371. and the expected major fragment ions at m/z=181. 146. and 145 (Figure 7a) . Product characterization: Peak 1 and peak 11 from Lil-Z) chromatography were shown to be OxIAA and MA. respectively. by the following procedures: peak I cochromatographed with authentic OxIAA on TLC with an R, of 0.2 using CHC1,:CH,OH:H;O 85:14:] as solvent. and had a retention volume on a 20 x 2.3 cm Lil-X) column of 92-1 10 ml identical to that of synthetic OxIAA: the HPLC retention volume on a Partisil 10 ODS 25 x 0.46 cm C.. column was 6.7 ml using We ethanol plus 1% acetic acid as solvent as was that of authentic OxIAA; and GLC of the pentafluorobenzyl ester gave a retention time of 9.3 minutes on a 1.8m x 2mm 1D 0V-17 column pro- grammed from Ill-250°C. which again was identical to the derivative of the authentic OxIAA. TLC of the diazomethane derivatized peak 1. developed in CHC 1,:Me0H:l-l,0 85:14:1, yielded the same compounds at R, 0.6 and 0.8 as did authentic OxIAA. GC-MS identified the compound at R, 0.8 as the methyl ester of OxIAA with a molecular ion at m/e=m5 and major fragment ions at m/z=146. 145. 128. and 117 (9). The compound at R, 0.6 was unstable to the normal conditions of GCoMS probably owing to lactam ring opening (18). Both the authentic and putative OxIAA yielded the same products and in similar proportions when treated with diazomethane. The putative OxIAA of Peak 1 was Ehmann negative (17) and the mass spectrum of the pentafluorobenzyl ester of peak 1 (Figure 7b) had a molecular ion at m/e=371 and characteristic fragment ions at m/z=181. 146. and 145 identical to those of authentic OxIAA (Figure 1a). Peak 11 cochromatographed with authentic 1AA on TLC with an R, of 0.42 using CHCl,:CH,OH:l-l.0 85:14:] as solvent. and was identical to 1AA on HPLC using 20% ethanol plus 1% acetic acid as eluant with a retention volume of 13 ml. Upon Lil-20 chromatography. the putative 1AA retention volume was 111-146 ml as was that of authentic 1AA. Peak 11 was Ehmann positive (17) and the GC-MS mass spectrum of the N-trimethylsilyl methyl ester had a molecular ion at m/e=%1 and characteristic fragment ions at m/z=X)2. 130. and 77. identifying it as 1AA. 100 28 75~ V-SOe 25~ 89 77 1-1.1. G IOO 35 YYITVTVlIT 557395 RELATIVE INTENSITY 75~ 75,3 ten :46 "T as 359 f“ In Bi an LTI‘Y 'IthI"; A‘UYTY TVTVETIVTIEYITYTYLIUAT‘ {7" IYIT ['I' l l I L I J l 1 us as 155 m m m 235 s 275 as m m 355 m as 181 I46 :45 369 "7,28 72 37: ii”. L l L L 115 1'35 155 175 1’5 1 m/z A. I A 11" ITITIYVYVII'FV 15235255275295 — 1'11 1 Al 1' Th 15 ETIEVII VIII] 5 395 Figure 7.--The 70 eV mass spectra of the pentafluorobenzyl ester of authentic oxindole-3-acetic acid (a), and that of the putative oxindole-B-acetic acid isolated from Zea_endosperm (b). 29 DISCUSSION By comparing the GLC peak area of the plant OxIAA to the peak area of an OxIAA standard of known concentration. it was estimated that 122 ug of plant OxIAA was obtained from the above purification pro- cedure. Since the yield from the purification procedure was about 6% with labeled authentic OxIAA. it could be calculated that 203 pg of OxIAA was present in 2500 corn kernels. This amounts to 387 pg per kg dry weight of kernel tissue. and would be about 170% as much as the free IAA in 4-day-germinated corn kernel tissue. Two findings suggest that OxIAA is a major catabolite of IAA. First. there is about as much OxIAA in the kernels as IAA. 387 ug-kg" (this paper) and 234 lag-kg" (7) respectively. Second. from the studies of Epstein et al. (7) on the turnover of IAA in the kernels. it can be calculated that in 4 h about one-half of the IAA of the kernel would be catabolized. We find that the labeled OxIAA isolated accounts for 26% of the expected IAA catabolized during the incubation period—and this does not include OxlAA that was further metabolized. Thus. this is the first demonstration that a major route of MA catabolism in corn kernels is oxidation of MA to OxIAA. 30 ACKNOWLEDGEMENT This work was supported by the metabolic Biology Section of the National Science Foundation. PCM M637. We thank Ms. P. Hall for assistance with use of the mass spectrometer MSU/NlH-’DOE facility sup- ported by PHS Rik-(1)480 and DE-AC02-768RO-1338. Technical assistance was provided by Ms. M. Urbano and Ms. C. Glaubiger and manuscript preparation was facilitated by Marianne LaHaine. This is journal article 9962 of the Michigan Agricultural Experiment Station. REFERENCES Ciesielski. T. (1872) Beitraege Biol. Pflanzen. 1. 1-30. Darwin. C.. and Darwin. F. (1880) The Power of Movement in Plants. Appleton. London. Heslop-Harrison. I. (1980) Plant Growth Substances 1979. F. Skoog editor. Springer-Verlag. New York. pp. 3—13. Bonner. 1.. and Thimann. KN. (1935) .I. General Physiol. 18. 649.658. Kogl. F. A.. Haagen-Smit. A. 1.. and Erxleben. H. (1934)Z. Physiol. Chem. 228:90-103. Hinman. R. l... and Lang. 1. (1965)Biochemistry 4. 144-157. Epstein. E. Cohen. J. D.. Bandurski. R. S. (1980) Plant Physiol. 65. 415-421. Davies. P. l. (1973) Physiol. Plant. 28. 954(1). Kinashi. H.. Suzuki. Y.. Takeuchi. S.. and Kawarada. A. (1976) Agr. Biol. Chem. 40. 2465-2470. Siehr. D. l. (1961)]. Am. Chem. Soc. 83. 2401-2402. Klambt. H. D. (1959) Naturwiss. 46. 649. Hall. P. I... and Bandurski. R. S. (1978) Plant Physiol. 61. 425429. Schlenlt H.. and Gellerman. 1. 1.. (1960) Anal. Chem. 32. 14124414. Epstein. E.. and Cohen. 1. D. (1981)). Chromatogr. 209. 413420. Martin. R. H. (1951) Nature 168. 32. Hinman. R. 1.... and Bauman. C. P. (1964”. Org. Chem. 29. 1N6.1215. Ehmann. A. (1977) J. Chromatogr. 132. 267-276. Julian. P.L.. Printy. H.D.. Ketcham. R.. and Doone. R. (1953) .I. Am. Chem. Soc. 75. 5305-5309. RESULTS Identification of Oxindole-B-acetic Acid The chromatographic similarities between authentic OxIAA and the isolated plant unknown I are summarized in Table l. The putative plant OxIAA and the authentic OxIAA had similar chromatographic properties on LH-20 lipophilic Sephadex, and TLC deve10ped with G solvent (chloroform:methanol:water 85:14:1) and A solvent (methyl ethyl ketone:ethyl acetate: ethanol:water 3:5:1:l). However, when either sample was reacted with diazomethane, a potent methylating agent, two zones of radioactivity were observed. Lengthening the reaction time of methylation had no effect on the relative amounts of the compounds. When the mixture was chromato- graphed on GLC only one peak was observed. GC-MS identified the compound as the methyl ester of OxIAA with a molecular ion at 205, and characteristic major fragment ions at 146, 145, 128, and 117. The mass spectral fragmentation pattern was identical to published spectra for the methyl ester of OxIAA (37), with the addition of an M-2 peak at 203. The two compounds could be separated by HPLC with 30% ethanol as eluant, after which the UV spectrum of the major compound with TLC R of 0.78, was found to be the same as OxIAA. The f 31 32 Table l.--Chromatographic similarities between authentic and putative OxIAA. OiKI’ATOGRAPHY PlANl mom 1 7 OXIMDLE-B-ACETIC ACID Lil-20 86-110 ML. . 92-1m ML. 0 SOLVENT 0.22 i 0.22 TLC " " (DIAZGVEfl-lAlE) 0.8, 0.78 i 0.6, 0.78 A SGMENT 0.83 i 0.8 HPLC FREE ACID 6—7 it. 7 ML. PFB ESTER 8 it. 8 it. GLC PFB ESTER 9.3 MIN. 9.3 MIN. 33 compound with R of 0.6 did not give a detectable GC peak f under the conditions employed, which precluded its identi— fication by GC-MS. The unidentified compound is most likely the methyl ester of the product of lactam ring opening (hydrolysis between the nitrogen and the carbonyl) which has been reported to occur under acidic (35, 44) and basic con- ditions (67), or the lactone ring formation (reaction of the side chain acid with the carbonyl) as suggested by Klambt to occur under basic conditions (39). On C18 HPLC the putative plant OxIAA and the authentic OxIAA cochromatographed both as free acids and as penta- fluorobenzyl (PFB) esters. Finally, by GLC both the PFB esters of the putative OxIAA and the authentic OxIAA had identical retention times. Final identification of the plant sample as OxIAA was accomplished by GC-MS. The PFB esters of both the putative OxIAA and the authentic OxIAA had identical 70 eV mass spectral patterns as are shown in Figure 7. As expected, the molecular ion of the PFB ester of OxIAA was at 371. An M-2 peak at 369 was also observed, which appears to result from dehydrogenation and rearrangement of the molecular ion to the stable quinolinium fragment. The methyl ester of OxIAA gave a similar M—2 peak, while the bis—trimethylsilyl (TMS) derivative of OxIAA had no M-2 peak. Since deriva- tization with the silylating agent bis-(trimethylsilyl)- trifluoroacetamide (BSTFA) derivatizes the side chain acid group and the nitrogen, a free N-H group appears to be 34 necessary for the M-2 rearrangement fragment. Other major fragments from the PFB esters were at mass 181, the penta— fluorobenzyl fragment, and at mass 146, and 145, the oxindole fragments. A summary of the major fragments of the PFB ester of the plant OxIAA and the authentic OxIAA is listed on Table 2. The percentage abundances between the plant OxIAA and the authentic OxIAA fragments agree very well. The rela- tive percentage abundances are, of course, dependent on the concentration of the sample analyzed, and the conditions of analysis, but the authentic and the putative samples varied similarly. Turnover of Labeled IAA In this study, 13% of the labeled IAA metabolized was converted to OxIAA after a 4 hour incubation period with endosperm tissue. The following discussion examines the data of Epstein et al. (17) to determine whether the turn- over observed in the present study approaches their reported value. Epstein et al. determined the first order rate constant for the dilution of specific activity of labeled IAA, when labeled IAA was added to the endosperm of 4 day old dark- grown Egg seedlings. The first order rate constant and the following rate equation can be used to calculate how much turnover of labeled free IAA would occur over a 4 hour incu— bation period: 35 Table 2.--Major GC-MS fragments and the percentage relative abundances for authentic and putative OxIAA. 7. ABUNDANCE m/z FRAGMENT PLANT OxIAA SAMPLE H 9 F F , 37' CI cazcocnz.r IO '3 N T) F F H 369 M-2 30 3: F F * l8l CH2.F IOO too F F H 146.145 ”CH2 ”CH2 72.57 83.59 ‘b N i) H 36 where: k = first order rate constant t = time CO = specific activity at time 0 Ct = specific activity at time t Epstein and coworkers calculated the first order rate con- stant to be 0.22/h. The specific activity of IAA at time 0 in the present experiments can be expressed as 0.32, for the ratio between labeled free IAA to total free IAA. Using this information and the rate equation, one may calculate C the specific activity of labeled free IAA t’ after a 4 hour incubation period in the endOSperm tissue. Ct was determined to be equal to 0.13; that is, 32% of the IAA is initially labeled at time 0, but only 13% of the IAA remains labeled after the 4 hour incubation owing to dilution by newly produced IAA. The total free IAA has been determined to remain rela— tively constant over these time intervals, so that the ratio of Ct to CO will equal the amount of labeled free IAA which remains unmetabolized after 4 hours: Ct _ IAA labeled at 4 h L IAA labeled at 0 h Co total IAA ° total IAA _ IAA labeled at 4 h — IAACIabeled at 03h g4%% or 41% of the labeled IAA remains unmetabolized. 37 If 41% of the labeled IAA was not turned over, then 59% of the labeled IAA would have been catabolized to other pro- ducts. Transport of labeled free IAA to other tissues and conjugation of IAA are not significant at this stage of development. The data of Epstein and coworkers, also found that 13% of IAA catabolized proceeds through decarboxylation, so that: 59% turnover — (59% turnover x 13% decarboxylation) = 51% turnover through carboxyl retaining catabolites Experimentally, the present study observed 13% turnover to carboxyl retaining IAA catabolites after.4 hours, or 26% of the expected rate predicted by the Epstein paper. The difference between the observed 13% turnover and the calcu- lated 51% turnover may be explained by differences in seed lot, isolation procedure, etc. The work of Epstein and coworkers predicted a rate of IAA turnover which approaches the level observed in this study, and they predicted a carboxyl retaining IAA catabolite which the present study confirmed by isolation and identification. Quantitation of Oxindole-B-acetic Acid in Zea mays Endosperm Tissue OxIAA (21.5 pg) with a specific activity of 10 pCi/umol was added to 1000 excised Egg kernels. After purification, the diluted final specific activity was determined to be 2.3 uCi/umol. This value was the average of two determi- nations, where the Specific activities were measured to be 38 2.27 pCi/pmol and 2.23 pCi/pmol. With these data and the isotOpe dilution equation, it was determined that there is 73 pg OxIAA/1000 kernels. Since there was 0.21 g dry wt/kernel, there was 356 pg OxIAA/kg dry wt. The amount of OxIAA determined from the feeding experiment, calculated using the external standard method, was 387 pg OxIAA/kg dry wt, and this agrees well with the isotope dilution value. The isotope dilution assay demonstrates that OxIAA is a natural component of £33 endosperm at a level near that of free IAA (17), 234 pg/kg dry wt. DISCUSSION The present work reports the first identification of OxIAA as a major carboxyl retaining IAA catabolite in higher plants. The amount of OxIAA present in 4 day old dark-grown endosperm tissue is 356 pg/kg dry wt. The most thorough prior identification of oxindole-3- acetic acid and dioxindole—3-acetic acid derivatives in plant tissues is from the work of Kinashi et al. (37). Using rice bran extracts, they identified the methyl esters of oxindole-3-acetic acid and dioxindole-3-acetic acid, their S-hydroxy analogues, and the free S-hydroxy-dioxindole- 3-acetic acid. Although Kinashi et al. pr0posed a route of IAA catabolism through oxindole-B-acetic acid and dioxindole- 3—acetic acid, the precursor-product relationship between IAA and these compounds was not demonstrated. Siehr (58) was the first to demonstrate that IAA or tryptamine may be metabolized to OxIAA by the basidiomycete HygrOphorus conicus. The present radiolOgical studies unambiguously demonstrate that OxIAA is a catabolic product of IAA metabolism in Zea endosperm tissue. 39 40 Oxindole-3-acetic Acid as a Major Route of IAA Catabolism There are several lines of evidence that indicate OxIAA formation is a major route of IAA catabolism in Zea_endo- sperm tissue. After a 4 hour incubation period, the extract contained only two peaks of radioactivity: unmetabolized IAA and OxIAA as isolated and identified by chromatographic and mass spectral analysis. IAA catabolic products which lost their labeled carboxyl group would not be identified by this isolation procedure. However, after the 4 hour incubation period, up to 90% of the initial radioactivity could be recovered by the 70% acetone extractions. Most of the remain- ing radioactivity could be accounted for in the endosperm tissue residue, so that catabolism through decarboxylation played only a minor role in IAA metabolism during the incu- bation period. This data confirms the work of Epstein and coworkers (17), who predicted that the majority of IAA catabolism proceeds through a carboxyl retaining route, since IAA was observed to turnover rapidly with low levels of IAA decarboxylation. Figure 2 indicates that there are two routes of cata- bolism in Zea endosperm tissue: a route with retention of the carboxyl moiety, and a route through oxidative decar- boxylation; nevertheless, there may be only a single route of catabolism of IAA, through oxidation of IAA to OxIAA with subsequent OxIAA decarboxylation. The metabolism of OxIAA must be further studied to resolve whether there are one or two pathways regulating IAA levels in Zea. 41 More evidence that OxIAA is a major catabolite of IAA metabolism is that the present study accounts for at least 26% of the predicted turnover of IAA to carboxyl retaining catabblites from the data of Epstein et al. (17). The exact proportions of IAA catabolism accounted for by OxIAA formation awaits complete turnover studies. Indole-3-acetic Acid Turnover Free IAA is turning over rapidly in the endosperm of 4 day old Zea endosperm tissue, but its relationship to the .growth of the seedling remains unknown. Free IAA does not appear to be an important transport form of IAA to the shoot or the root (25); but IAA conjugates, as IAA inositol, do appear to be IAA precursors for vegetative growth (49). When [3H]-IAA is applied to the cut endosperm of Zea seed- lings (25), 98% of the radioactivity observed in the shoot after 8 hours of incubation is no longer IAA or IAA esters. The identity of the IAA metabolized during transport is unknown. At this stage of development, IAA is not being con- jugated in the endosperm, so the purpose of the high turn- over rate of IAA is unknown. The turnover of free IAA may be a way of regulating the levels of IAA in endosperm tissue, since the majority of free IAA is not being transported to actively growing vegetative tissue. Corn endosperm tissue is generally considered to be storage tissue surrounded by the living aleurone layer, scutellum, and embryo (36). The presence of free IAA and its catabolism in endosperm tissue 42 may indicate a role of IAA in seedling development during germination. Biological Activity of Oxindole-B-acetic Acid After Hinman and Bauman (30) developed a new synthesis for OxIAA, several groups tested the biological activity of OxIAA. Galston and Chen (22) reported the OxIAA had bio- lOgical activity with etiolated, and green pea stem sections. The greatest increase in growth occurred between the concen— 4M for green pea sections, and between 4 3 trations 1.6-5.2 x 10- -l.6 x 10- M for etiolated pea sections. The growth promotion induced by 1.6 x lO-4M OxIAA the concentrations 1.6 x 10- was reported to be similar to the growth promotion with 2 x 10-4 M IAA in green pea sections. All subsequent research- ers have shown that OxIAA is inactive in growth promotion in the bioassays tested. Weis (69) observed that OxIAA did not promote growth of tobacco tissue cultures, and 4 7 inhibited fresh weight increases at 10. -10- M. OxIAA was inactive in the Avena first internode test, and the Avena coleoptile curvature test in the concentration range 5 x 10-8 to 5 x 10-4 M (28). Kinashi et al. (37) found no increased _growth in A3323 coleOptile sections when treated with 0.1 to 100 pg/ml for the methyl esters of OxIAA, S-hydroxy-OxIAA, dioxindole-B-acetic acid, or S-hydroxy—dioxindole-3-acetic acid. The same group (63) showed that 5-hydroxy-dioxindole- 3-acetic acid, and 5-hydroxy—OxIAA were synergistic in IAA promotion of ethylene in mung bean hypocotyl segments, but 43 OxIAA and dioxindole-3-acetic acid were not, in the concen- tration range 0.1-100 pg/ml or 5.2 x 10'7-5.2 x lo‘SM OxIAA. The general concensus is that OxIAA does not promote growth in the bioassays tested. The work of Galston and Chen should be repeated to show whether OxIAA is growth pro- moting for pea stem sections, is metabolized to other growth promoting substances, or was contaminated by a product of OxIAA synthesis from IAA. Galston and Chen used TLC and melting point analysis as an indicator of OxIAA purity. Assuming that OxIAA is inactive as a growth regulator in corn, it is interesting to speculate that the catabolism of IAA to OxIAA occurs when IAA is "used up" during growth. Other possible carboxyl retaining IAA catabolites: zeanic acid, 2,8 dihydroxycinchonic acid, isolated from corn steep liquor (47), and B-acid, 2,6 dihydroxycinchonic acid, isolated from rice bran (37), (Figure 8), have been reported to have biological activity. Zeanic acid was reported to increase grape fruit set, and to promote dwarf rice seedling growth. It was also reported to increase proliferation of rice callus growth, and radish cotyledon growth in the absence of kinetin and auxin (48). B-acid was observed to supress dwarf rice seedling growth (37). However, when OxIAA and dioxindole-B-acetic acid are isolated under simi- lar acidic and basic conditions, ring expansion to similar oxo-quinoline-4-carboxylic acids are observed (35, 67), (Figure 9). Until zeanic acid and B-acid are isolated under 44 COOH COOH HO H \\\\ ‘E_-C) \\\~ -—+> /// OH c N N 0 B-ocid H COOH COOH \ (— \ //' ._9 OH 5 N [u 0 OH OH H Zeanic acid Figure 8.--The keto and enol forms of zeanic acid and B—acid. 45 A.cofiumeHouomumno nonunom muflw3m «de0 mo posooum homeomcmunmmn nonmamumo omen 029V .oflom oanoUMImtoHOpConwp one <