a E 00‘». at”. .- {IIXOthO Q.Oo\4~ I Iv. Dub... . . 3. In E i; m??? if . I ‘0 -‘ 1‘1.v I... 1.o.¢-!!..J .l..: .A ? «ouch... o. “I . r Ilaxu v». . .1 “".'\I :4“ 19'. 'x ..‘avovy't.‘.vf..4ytt.n vol-VII...I oIA(-I 0., 0‘1 '9 i pong. .‘v. ov.«..c7de o . 322.1 907. . ‘1:r-- . v I I. . . .. a In. 1. «If:- lflfi’llhih , . . . '19:. >1. r . 7 .. . . . . >0. v.. x .. 7.....‘f1c‘t‘l . . I ..c.... ‘ N'- . v | .. . .5.... at i. . - w . . . .. . . _ . . . EVE??? . .. v . :tl. s-vtfiovu.’ It}- .I . av...§.§adh. .. - .......... . . . . . .. . ILO.\I.I.UO ....IVO. vli . .. .435... \ This is to certify that the dissertation entitled THE BIOSYNTHESIS 0F INDOLE-B-ACETIC ACID IN g5 MAYS presented by PHILIP JOHN JENSEN has been accepted towards fulfillment of the requirements for Ph D degree in BO ta HY (Plant Physioloqv) act 3 ~ Esau... it Major professor Date k)“ it.) J I ’94 Z—r MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 ..._.__.. fi fi - -r # fl 4....— '— r fl “DIARY Mun Cute 4 University \ - J PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE .oqloo j __ -—————-—— w I usu le An Affirmative ActlorVEquel Opportunlty Institution ottoman”! THE BIOSYNTHESIS OF INDOLE-3-ACETIC ACID IN ZEAAbflS BY Philip John Jensen 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 1992 ABSTRACT THE METABOLISM OF INDOLE-B-ACETIC ACID IN ZEA HAYS BY Philip J. Jensen The onset of de novo synthesis of indole-B-acetic (IAA) and tryptophan in seedlings of Zea mays was examined using deuterium labeled water as a general precursor. IAA and tryptophan synthesized via the shikimic acid pathway would contain deuterium in one or more positions. We have demonstrated, by GC-Ms-SIM, that 7 day old, dark grown seedlings grown on 30% 020 incorporate deuterium into tryptophan, but not into IAA. Protium and deuterium NMR spectra indicate at least the six position in the ring structure of tryptophan is labelled. Kernels of Zea mays accumulate large amounts of IAA during development. A single endosperm may contain as much as 0.5 umol of esterified IAA. The origin of the IAA has never been demonstrated. To answer that question GC-MS assays of free IAA and ester IAA were made throughout the course of development. Ester IAA appears at day 10 and at day 45 reaches a maximum value of 0.5 pmol kernel-1. Free IAA never accumulates so we conclude that either IAA biosynthesis in the kernel or transport of IAA esters, and not esterification, is the rate limiting step. Zea mays endosperm cells can grow in vitro on media containing no growth hormones. This suggests the endosperm is the site of IAA biosynthesis in the developing kernel. A time course study using endosperm cells in suspension culture has provide clear evidence that these cells are indeed synthesizing IAA. There was an increase of nearly 10 pg of total IAA per 20 gm fwt. in 6 days. This system will be ideal for studying the biosynthesis of IAA and its regulation. The endosperm of Zea mays most likely contains all the enzymes required to biosynthesize IAA de novo. To determine the feasibility of this system for examining the ability of putative IAA precursors to sustain IAA synthesis we have examined the conversion of’lAC-anthranilic acid to IAA in isolated endosperm. Anthranilic acid was used because it is common to all proposed pathways yet occurs early enough so as not to presuppose one pathway over another. Data relating to the rate of IAA synthesis from anthranilic acid and proof as to the identity of the IAA will be presented. ACKNOWLEDGMENTS I first would like to acknowledge Dr. Robert Bandurski for giving me the opportunity to work in his lab and the freedom to pursue the questions that intrigued me. I am also grateful to Aga and Mark for their help. Also of great enjoyment was my exposure to the visiting scientist, Toni, Stanley, Jacek, Maciej, and Natalya, who opened my eyes to other countries. I would like to thank my parents, Mom and Dad, for instilling in me an interest in science and their confidence in my abilities. Finally I would like to thank my therapeutic aide, Carolyn, who is more important than any degree, for her support and companionship. IV TABLE OF CONTENTS Page LISTOF TABLESOOOOOOOOOOO0.0.0.000...OOOOOCOOOOOOOOOOOOOVIII LISTOF FIGURES.00......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCIX LISTOFABBREVIATIONS...0.0.0.0000...OOOOOOOOOOOOOOOOOOOOOXI INTRODUCTION. 0 O O O O O O O O I O O O O O O O O O O O O O ..... O O ...... O O O O O O O O O O 1 LITERATURE REVIEW ............. . ......... ...... ..... ........1 A O HORMONES AND GERMINATION O O I O O O O O O O O O O O O O O O C O O O I O O O O O O O O 1 B. C. D. E. Function and origin of hormones during germination...2 Role of auxins in dormancy and germination...........4 Auxin dynamics during germination....................7 Metabolism and transport of IAA during germination...8 The origin of building blocks during germination....12 The transition from heterotrophy to autography......14 DEUTERIUM EFFECTS ON BIOLOGICAL SYSTEMS...............16 INDOLE-3-ACETIC ACID BIOSYNTHESIS IN DEVELOPING SEEDS.24 AUXIN DYNAMICS DURING SEED DEVELOPMENT................28 INDOLE-3-ACETIC ACID METABOLISM IN TISSUE CULTURES....34 INDOLE-3-ACETIC ACID BIOSYNTHESIS RESEARCH WHERE IS ITHEADED?‘.OOCOCOCOOCCCOOOCO0.0.0.00000000047 Proposed biosynthetic pathways......................48 Steps toward definitive answers.....................56 LITERATURE CITED.OOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0000000000064 EXPmIMENTALOOOOOOOO ...... OOOOOOOOOOOOOO ..... 0.0.0.000000084 EXPERIMENTAL IOOOOOOOOOIOIOOOOOOO ........ 0.0.0.00000084 INCORPORATION OF DEUTERIUM INTO INDOLE-B-ACETIC ACID AND TRYPTOPHAN IN 25.4mm SEEDLINGS GROWN ON 30% 020 ABSTRACTOOOOOOOOOOOOOOOOOOOOO0.0.0.000000000085 INTRODUCTIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.086 MATERIALS ANDMETHODSOOOOOOOOO0.0.0.00000000087 Purification of indole-3-acetic acid and tryptophan............................88 GC-MS analYSiSOOOOOOOOOOIOOOO0.0.0.00000091 RESULTS AND DISCUSSION.......................92 LITERATURE CITED............. ........ .......109 EXPERIMENTAL 1100.......00..0.00.00...00.000.000.000111 METABOLISM OF INDOL-B-YLACETATE IN MATURING KERNELS OF ZEAADHG ABSTRACTOOOO ......... O OOOOOOOOOOOOOO 0.0.0.00112 INTRODUCTIONOOOOOOOOO0.0... ..... O. ..... 0.00.113 MATERIALS AND METHODS.......................ll4 Plant material..........................114 IAA purification........................115 GC-MS-SIM analysis....... ..... ..........117 RESULTS............ ..... ....................117 DISCUSSION........... ..... ..................125 LITERATURE CITEDOOOOOO...0.0.000000000000000131 VI EXPERIMENTAL IIIOOOOOOO...O...OOOOOOOOOOOOOOOOOOO00.134 METABOLISM OF INDOL-B-YLACETATE IN ENDOSPERM SUSPENSION CULTURES OF ZEAMdYS ABSTRAflOOOOOOOOOOOOOOOOOOOOOOOOOO00.0.0.0..135 INTRODUCTIONOOOOIIOOI ...... 0.0.00.0000000000136 MATERIALS AND METHODS.......................138 Plant material..........................138 1AApurificatiODOOOIOOOOOOOOO0.0.0.0....138 GC-MS-SIM anaIYSiSOOOOOOOO0.0.0.00000000140 RESULTS AND DISCUSSIONOOOOOOOOOOOOOOO00.0.00140 LITERATURE CITED............................149 EXPERIMENTAL IVOOOOOOOOOOOOOOOOOOOOOOOIO00.000.00.00152 ISOLATED IMMATURE ZEAADHG ENDOSPERM AS A MODEL SYSTEM FOR STUDYING INDOLE-3-ACETIC ACID METABOLISM ABSTRACTCOOOOOOOOOOOOOOOOOOOOOOO0.0.00.00000153 INTRODUCTIONOOOOOOOOOOOOO ..... 0.0.00.0000000154 MATERIALS AND METHODS.......................157 Plant material..........................158 IAA purification....... ...... ...........159 GC-MS analysis..........................159 RESULTSOOOOOOOOOOOOOOOOOOOOOO0.0.0.0.0000000159 DISCUSSION. ......... .. ............... .......166 LITERATURE CITEDOOOOCOOOOOOO00....00.0.0000000000000169 ISOLATED IMMATURE ZEAADHT ENDOSPERM AS A MODEL SYSTEM FOR STUDYING INDOLE-B-ACETIC ACID METABOLISM ABSTRACTOOOOOOOOOOOOOOOO0.0.00.0000000000000153 INTRODUflIONOI0.0.0.000...00.0.0000000000000154 MATERIALS AND METHODS.......................157 Plant material..........................158 VII IAA purification........................159 GC-MS analy31800000000000000.0.000000000159 RESULTS.....................................159 DISCUSSION..................................166 APPENDICES..........................................l7l APPENDIX A..........................................172 Amide IAA Content in Kernels of Zea mays var. Silver Queen 0 APPENDIX B0.00...OOOOOOOOOOOOOOOOOO0.000000000000000174 Affect of the Kernel on Cob Development in Zea mays. VIII List Of Tables Table Page INTRODUCTION Table 1. Amounts Of free and conjugated IAA in various seeds and other plant tissues (from Bandurski and Schulze, 1977).............................5 Table 2. A summary of the structures and amounts Of indole-B-acetic acid and its conjugates in Zea mays kernBISOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.9 Table 3. Deuterium effects reported in plants. ....... ..19 Table 4. IAA levels reported in culture ...... ..... ..... 36 Table 5. Max. IAA levels reported during culture cycle.37 EXPERIMENTAL 1 Table 1. Incorporation of deuterium into indole-3-acetic acid and tryptophan by Zea mays grown on Hzo or 30% D20 for 7 days in the dark...............103 Table 2. Percent abundancies of the isotope peaks for the MS fragment at m/z 130 for D3 2', 3', 3', Tryptophan before and after base exchange treatment....................................104 Table 3. Percent abundancies of the isotope peaks for the MS fragment at m/z 77 for D3 2', 3', 3', tryptophan showing movement Of side chain deuterium into the benzene ring fragment.....104 EXPERIMENTAL 4 Table 1. DPM recovered in IAA fraction after HPLC purification step. IAA was isolated f om Zea mays endosperm after incubation with 1<}- anthranilic aCidOO00.00.000.000.00.0.00000000161 Table 2. Ratio Of 14C to 3H in IAA fraction at various stages of purification... ..... ..... ..... .....163 IX Figure List of Figures Page INTRODUCTION Figure 1. Figure 2. Figure 3. Proposed pathways for indole-3-acetic acid biosynthesis in plants........................49 IAA biosynthetic pathway variations and regu1ation mecnanismSOOOOOOOOOIOO0......0.....54 Proposed alternate pathways for indole-3-acetic aCid biosyntheSiSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeS? EXPERIMENTAL 1 Figure 1. Figure 2. Figure 3. Mass spectral fragmentation patterns for methly- ated IAA (A) and methylated and acetylated tryptophan (B)0.0.0..0.00.00.00.000.00.00.00.09? GC-MS total ion chromatograms of methylated IAA (A) and methylated and acetylated tryptophan (B) isolated from Zea mays seedlings grown in 30% 020 for 7 days in the dark....................99 Partial mass spectrum showing the quinolinium ion and its associated isotope peaks from control (A) and methylated and acetylated tryptophan (B) isolated from Zea mays seedlings grown in 30% 020 for 7 days in the dark. An increase in isotope..........................101 Figure 4A. 1H-NMR spectrum Of methylated and acetylated tryptophan isolated from Zea mays seedlings grown in 30% 020 for 7 days in the dark. ..... 105 Figure 4B. 2H-NMR spectrum of methylated and acetylated tryptophan isolated from Zea mays seedlings grown in 30% 020 for 7 days in the dark. .....107 EXPERIMENTAL 2 Figure 1. Comparison of changes in the levels of free and total IAA (A) and the accumulation Of fresh and dry weight (B) during the growth and maturation of maize kernels. Vertical bars indicate the standard error from the mean of four replicate samples.................. .......... .. ....... .119 Figure 2. Comparison Of changes in the levels of free and total IAA when calculated on a per gram dry weight basis (A) and a per gram fresh weight (B) basis during the growth and maturation of maize kernels. Vertical bars indicate the standard error f......................................121 Figure 3. Changes in the levels of free IAA in the kernel during development...........................123 Figure 4. Percent of total IAA in the ester form during kernel development. The remaining IAA is in the free aCid fomeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee124 EXPERIMENTAL 3 Figure 1. Fresh and dry weight Of Zea mays endosperm cultures during the culture cycle. Culture were inoculated at a high density to obtain a rapid growth rate..................................145 Figure 2. Comparison of changes in the levels Of free and total IAA (A) and the accumulation of fresh and dry weight (8) during the life cycle of Zea mays endosperm suspension cells. Vertical bars indicate the standard error from the mean....146 EXPERIMENTAL 4 Figure 1. Time course showing DPM associated with IAA £faction from isolated endosperm incubated with C-anthranilic aCid. O O O O ...... O O O O O O O O O O O O O O 162 Figure 2. (A) GC-MS chromatogram Of HPLC fraction containing methylated IAA urified from isolated endosperm incubated with 1 C-anthranilic acid. (B) Mass spectrum of single peak Observed in GC- MS chromatogram. (C) Mass spectrum Of a methylated IAA ...... . ........................ 164 APPENDICES Figure l. (A). Cob development after removal Of a 6 X 12 block Of kernels from the ears five days after pollination and (B) subsequent application of 1% IAA in lanolin (w/w).........................l76 XI 14C DAP 2}! DEAF GC-MS GC-MS-SIM HPLC IAld IAA IPyA 2,4-D TNH2 TRP ABBREVIATIONS carbon 14 days after pollination deuterium diethylaminoethyl gas chromatography-mass spectrometry gas chromatography-mass spectrometry-selected ion monitoring high pressure liquid chromatography indole-B-acetaldehyde indole-3-acetic acid indole-3-pyruvic acid mass to charge ratio mega Hertz nitrogen 15 nuclear magnetic resonance 2,4-dichlorophenoxy acetic acid tritium tryptamine tryptophan XII 1 HORMONES AND GERMINATION Webster's New World Dictionary defines germinate as: 1) to sprout or cause to sprout, as from a spore, seed or bud, 2) to start developing or growing. Among plant scientist there is a debate over the definition Of germination which revolves around the question of when germination ends. All would agree that as soon as the radicle has emerged the seed can be considered germinated; but the endpoint of germina- tion is unclear. In general, germination has been divided into two phases (Berrie and Drennan, 1971, Sen and Osborne, 1974). Phase I includes those events leading up to the emergence Of the radicle. Desiccation during this stage does not result in the death Of the seed. Phase II includes the subsequent events relating to seed reserve dependent seedling growth. Desiccation during this phase results in death. Based on these phases, germination ends when the seedling becomes au- totrophic. The majority Of research done on hormone rela- tions in germinating seeds involves only phase I. There is a conspicuous lack Of data during phase II Of germination. In contrast, the growth of cereal seedlings has been divided into three stages (Derwyn awd., 1966) Stage I con- sists Of the heterotrophic period beginning with the imbibi- tion Of water and ending with the commencement of photosyn- thesis. Stage II is the transition period when hydrolysis Of reserves and photosynthesis occur simultaneously. Stage III is the autotrophic period when the seedling is totally reliant on photosynthesis for its nutrition. Function and origin of hormones during germination During the stages Of germination, extensive elongation occurs. This elongation must be under hormonal control. Seeds are very rich sources Of plant growth substances (Bandurski and Schulze, 1977). The role Of these compounds in germination is not well understood. Most of the research done in this area has concentrated on the release Of seeds from dormancy and phase I Of germination. The best under- stood hormonal aspect Of germination is the promotion Of a- amylase production in the aleurone cells of cereals in re- sponse to gibberellins (GA) released by the embryo. Addi- tional research has shown exogenously applied GAs can pro- mote germination in dormant seeds. Though it is known that GA breaks dormancy, the exact mechanism is still uncertain. There is only a limited amount Of evidence however showing that GAs are synthesized during exposure tO dormancy break- ing treatments (Jones and Stoddart, 1977). During germina- tion, in contrast, the demmo biosynthesis of GAs during ger- mination has clearly been demonstrated (Jones and Stoddart, 1977). Abscisic acid (ABA) is widely believed to be responsi- ble for seed dormancy. ABA appears to act by suppressing the production Of specific enzymes while inducing the pro- duction of others (HO add. 1989). In cereals, ABA is in- volved in the prevention Of precocious germination but is not involved in maintaining dormancy (for review see Zeevart and Creelman, 1988). During germination the only putative role for ABA is acting as an antagonist Of GA promoted events during germination (Walton, 1977, Chrispeels and Varner, 1967b, Khan and Downing, 1968). Cytokinins are best known for their role in differenti- ation Of callus tissue in culture. They have also been show to function during germination. Cytokinins can affect the permeability of membranes, thus regulating the release Of GAs from the germinating cereal embryo (Thomas, awd., 1975). Cytokinins also play a role in the transcription and trans- lation Of DNA but a mechanism for this has not been eluci- dated. Stimulation Of ethylene production by cytokinins has been proposed as a factor in bringing about germination in dormant peanuts (Ketring and Morgan, 1971). Ethylene can break dormancy in many seeds including clover, cockleburr, lettuce and celery (Ketring, 1977). In the case Of the peanut, the ethylene involved in dormancy breaking is believed to be synthesized in the embryo (Ketring and Morgan, 1969) During growth Of etiolated peas ethylene appears to act as an inhibitor Of elongation in re- sponse to an increase in auxin levels. Auxin itself is not an inhibitor Of elongation in peas (Burg and Burg, 1966). In etiolated corn and oat coleoptiles, ethylene production does not continuously increase with increasing auxin concen- trations as in etiolated peas. Ethylene formation and growth reached maximum rates around 10'5 M IAA and declined as more auxin was added (Burg and Burg, 1966). Luckwill (1952) was the first to suggest that dormancy is regulated by a balance of endogenous inhibitors and pro- moters. Cytokinin application can overcome dormancy induced by exogenous abscisic acid (ABA) (Aspinal awd., 1967, Khan, 1967). In contrast, GA cannot overcome this induced dor- mancy (Khan and Waters, 1969). Recently, Karssen and Lacka (1985) , working with GA and ABA mutants of Arabidopsis thaliana, concluded the two hormones never act simultaneously in the seed. ABA induces dormancy during seed development and GA stimulates germination. This is in contrast to the conclu- sions of Walton (1977) but may represent a real difference between Arabidopsis and cereals. Role of auxins in dormancy and germination Seeds contain abundant stores of IAA (Table 1). Even though it is present in large amounts, it is generally be- lieved that IAA plays an insignificant role in the breaking of dormancy. To date only Nikolaeva (1970), working with Aav, and Sircar (1967), working with rice, have presented data suggestive of a role for IAA in the physio- logical events leading to the induction of germination. Both investigators theorized that high IAA Table 1. Amounts of free and conjugated IAA in variuos seeds and other plant tissues (from Bandurski and Schulze, 1977). Species Tissue 1AA Content Free lAA' Ester-[AAh Amide-IAAc rig/kg" ing/kg" rig/kg" Cereals . Avena saliva Vegetative tissue 16 5 69 Aoena sativa Seed 440 7,620 n.d.f Hordeum vulgare Seed (milled) 40‘ 329 --‘ Oryza saliva Seed 1,703 2,739 — Panicum miliaceum Seed 633 3,198 — Triticwn aestivum . Seed 123 511 — Zea mays Vegetative tissue 24 328 60 Zea mays Seed 500 to 71,600 to — 1,000 78,500 Legumes Glycine max Seed 4 50c 524 Phaseolus vulgaris Seed 20c 30c 136 Pisum sativum Vegetative tissue 35 5 43 Pinon sativum Seed 93 n.d. 202 Others Cocos nucifera Liquid endosperm 0 905 — Fagopyrwn csculentum Seed 40 127 25 Helianthus annuus Swd 30r 110f — Lycopersicwn exculentum Fruit Trace Trace — Saccharomyces cerevisea Packed cells 290 n.d. - ' No alkaline hydrolysis. " IAA after hydrolysis with 1 N alkali minus the free IAA. ‘ IAA after hydrolysis with 7 N alkali minus the free and ester IAA. " Seedlings and fruits are fresh weight, seeds are air dry and yeast cells contain 30% dry matter. ‘ A visual estimate of IAA was made on a TLC plate since calorimetry was precluded by contaminants. ‘ n.d. (not detectible). Where the ester content is high, small amounts of IAA escape detection. ‘ A dash (—) indicates the assay was not done. levels inhibit germination and only after these levels have decreased can the seeds germinate. Once dormancy has been broken, the embryo can begin germination. In Zea mays the initial step of germination is cell elongation, which is then followed by cell division (for review see Berlyn, 1972). Since elongation occurs first it is reasonable to assume IAA is required during the initial stages of germination. In addition to its well known and studied role in elon- gation, IAA has also been suggested as having some function in regulating a-amylase levels during germination . MacLeOd and Palmer (1969) found that IAA can stimulate a-amylase formation when applied 12 hours prior to adding GA. No stimulation was found when the two hormones were applied simultaneously. Detipping of the coleoptile also leads to a reduction in a-amylase production. Working under the assum— tion the coleoptile tip was the site of auxin production ad- dition of IAA to detipped coleoptiles resulted in normal levels of a-amylase, confirming their hypothesis. Verbeek awd. (1973) found IAA could reverse the decrease of a-amy- lase activity induced by kinetin. They also found a corre- lation between a-amylase activity and coleoptile length as both reach their maximums at the same time. 7 Auxin dynamics during germination Perhaps the best physiological study of IAA levels and the various events occurring during germination has been done by Sircar (1967) working with rice. Immediately after imbibition, free auxin levels increase in both scutellum and endosperm. Total auxin levels were not measured. Auxin in the endosperm adjacent to the scutellum disappears first. This disappearance is under the control of the embryo. When the embryo and epithelial layer were removed after sprout- ing, there was no further decrease in IAA in the endosperm. Free IAA levels increase for the first 72 hours in both the embryo and endosperm as does the availability of respirable materials, including amino acids. After 72 hours, there is a general decline in the levels of all these compounds. As- paragine is the predominant amino acid in the endosperm af- ter 24 hours. By 48 hours the four amino acids, arginine, tyrosine, ornithine and tryptophan, which are all absent in the endosperm, arise by transamination in the embryo. They believe the increase of tryptophan between 48 and 96 hours to be due to synthesis from serine condensed with indole. The indole moiety was believed to have come from IAA which disappears during germination. The most complete biochemical study of IAA levels dur- ing gemination has been done on Zazmqmu Ueda and Bandurski (1969) determined not only the levels of free IAA during germination but also the levels of ester IAA, including 1-0 and 2-0 indole-3-acetyl-nwo-inositol, and their arabino- sides. All of the various IAA esters declined at a rela- tively uniform rate during germination. The total amount of IAA in the whole seedling decreased nearly 90% in the first 96 hours of germination. Free IAA levels rose during the first 24 hours and then declined at a constant rate. During this study, the occurrence of five new IAA compounds was also shown. The breakdown of IAA compounds in Zea mays ker- nels is shown in Table 2. Metabolism and transport of IAA during germination Free IAA and its potential precursor tryptophan are not transported from the seed in sufficient quantities to sat- isfy the needs of the shoot (Hall and Bandurski, 1978, Nowacki and Bandurski, 1980). Indole-3-acetyl-nwo-inositol (IAInos) is the auxin transported from the seed to the shoot in Zea mays (Nowacki and Bandurski, 1980, Komoszynski and Bandurski, 1986, Chisnell and Bandurski, 1988). Also working with thmamy Whitehouse and Zalik (1967) concluded free IAA was not transported from seed to shoot. Additionally, they found that tryptophan is not actively transported through tissue segments. This agrees with the findings of Schrank and Murrie (1962) who found that trypto- phan is not translocated to any great extent in oat coleop- tiles. Jackson and McWha (1983) have found that like Table 2. A summary of the structures and amounts of indole- 3-acetic acid and its conjugates in Zea mays kernels. 1C) <<~ can mu_ca «moon—o cm on a nm.~m o.mm gc.r coo=_o o_oo_=_1oo e..i s roo=_s-1_zzoo.-m-.iooc_v ro.~v ~.~m 4,o so -cé-23:339.Eotoirn-rim fl..Hfller\m zm.ilbtox 11%V\\\)vcx .( . o u~.n~ v.m~ o\\\/LJ www.moc_notc-—ouwmocmPsumuco_ouc_ A... a.” .ozaooc.-osr-1.5zoo.-n-o_ocriv-bo-_ um.o_ 0.“ x zoos/Inn“. ox .oummocwresr-aqueuorn-m_oe:_v.c-~ 8 u~.m_ ".oa mflwumiWHxth,x~ Irrexor «Foaimcc._5coooo_ouc_ x .....w.o m.o \ x o twuo uvuoUOonim—ODC— fix _.|/_.h,¥re. mxuoz 4ao z. pznox< manhunzpm ozaoazou Illl iilr‘ ii 11 inLZM, free IAA is not transported from seed to shoot in Avena seedlings . In a study of 4 day Old Zea mays seedlings, Epstein etal. (1980) found that IAA in the seed is rapidly turning over, most being metabolized to unidentified products. Free IAA appears in the endosperm through ester hydrolysis, not through synthesis from some precursor. The IAArwwo-inosi- tols however turn over more rapidly than the rate at which free IAA appears. It is possible that free IAA is being re- leased from an IAA-glucan and not IAArwwo-inositols (Piskornik and Bandurski, 1972). It was postulated that the IAArwwo-inositol glycosides play a role in glycosylation and/or in regulating the concentration of IAArwvo-inositol. Pengelly and Bandurski (1983) studied the IAA metabolism in Zea mays seedlings using deuterium oxide as a tracer. Their data indicated that IAA was being made de novo in 4 day old light or dark grown seedlings. The de novo biosynthesis accounted for 25% and 9.6% Of the IAA in the shoots and roots respectively. The remainder is from seed reserves. The IAA of the shoots was labeled at a greater rate suggestive of different biosynthetic pathways operating in shoots and roots. In beans, seed stores appears to play a minor role in supplying the developing seedlings with IAA (Bialek and Cohen, 1991) The IAA required for growth is ap- parently synthesized.demmm as germination proceeds. This 12 may represent a fundeamental difference in IAA metabolism between corn and beans, and possibly monocots and dicots. The origin of building blocks during germination During germination the food reserves stored in the seed begin to break down. Hydrolysis of these reserves provide the seedling with the nutrients required for growth. The compounds released may be used directly to build new macro- molecules, Or may be further metabolized and serve as pre- cursors for other compounds. There exists great variation among seeds in the mechanisms and timing of these events. These differences are especially evident when comparing monocots, dicots, and gymnosperms. In some dicot seeds the cotyledon is the major storage tissue, in others it is the endosperm. Additionally the major storage compound can be starch, protein or lipid. All of these factors affect the synthesis of new compounds. The starch stored in the endosperm of cereals is broken down to hexoses which are transported into the scutellum where they are converted to sucrose (James, 1940) for trans- port throughout the seedling. The pentose phosphate pathway in particular is very active in metabolizing carbohydrates in germinating seeds. This pathway provides the embryo with NADPH (NADH is provided via glycolysis and the TCA cycle)(Roberts, 1969). The pentose phosphate pathway also 13 produces precursors of the aromatic amino acids and lignin (Higuchi and Shimada, 1967) Hydrolysis of storage proteins results in the release of amino acids. The metabolic fates of these amino acids vary. Most are transported intact to the seedling (Oaks, 1965). Others, including glutamic, aspartic, their corre- sponding amides, and proline are extensively metabolized (Oaks, 1965). The amino acids in the fat storing seed of castor bean are extensively used in gluconeogenesis (Stewart and Beevers, 1967). The amino acid metabolism during germi- nation in corn differs from that in oats and barley. When removed from their food source, embryos of eat and barely grow feebly (Harris, 1954) while those of corn appear normal (Dure, 1960). This may represent a difference in capacity for'demme synthesis of amino acids. Oaks and Beevers (1964) found that while the corn embryo is capable of synthesizing amino acids, the supply of organic nitrogen from the en- dosperm in some way curtails endogenous synthesis within the embryo. Excised embryos were able to synthesize glutamic and aspartic acids from glucose and inorganic nitrogen. Synthesis therefore appears to occur when seed supplies are not sufficient to meet the demands of the seedling. Thus the rate limiting sequence of reactions are those involved in the further conversion of these key amino acids. They also reported that tryptophan levels were low, while leucine and proline were present in large amounts suggestive that 14 zein is the principle donor of amino acids. This suggests the possibility that low reserves of tryptophan in corn may enhance its de novo synthesis at a comparatively young age with respect to the other amino acids. The biosynthesis of amino acids in 6 day old barley seedlings has been studied by Mitra awn. (1976) using deuterium oxide. All of the amino acids studied, including phenylalanine and tyrosine, became deuterated to some extent indicating their biosynthe— sis. This indicates that by six days of age the seed can no longer meet the amino acid demands of the seedling. Nucleic acids and nucleotides stored in the endosperm and scutellum of the corn kernel are of minor importance as a reserve for the developing embryo (Ingle and Hageman, 1965). Synthesis of nucleotide material therefore most likely occurs in the developing embryo. The transition from heterotrophy to autotropy At some time the seedling must switch from a het- erotrophic existence to an autotrophic one. The age at which this occurs varies not only with the type of plant studied but also with the compound being studied. Some com- pounds are synthesized demmo immediately at the beginning of germination, others are not synthesized.demmm until the seed reserves have been depleted. The transition to autotrophy in Zea mays seedlings with respect to overall carbon metabolism has been determined by Deleens awfl. (1983, 1984). 15 Using C02 depleted.iril3c they found seedlings to be totally dependant on seed carbon reserves for the first 7 days. Af- ter 7 days, carbon from photosynthesis begins to appear. The seed continues to supply carbon to the seedling for up to 4 weeks. The autotrophic carbon is preferentially used for respiration while carbon from the seed is used for building leaf matter. The age of transition was also shown by observing the enzyme parameters of phosphoenolpyruvate carboxylase which change depending on the carbon source. Very little is currently known about the transition to autotrophy with respect to tryptophan, IAA, or the other plant hormones. This is undoubtably one of the most impor— tant events during a plant's life. Failure to establish a self sufficient nutrition will ultimately result in death. Despite the importance of this transition little is known about the timing, signals, etc. that bring about this change. Altering IAA metabolism during this critical period may prove to be an effective means of regulating the subse- quent growth of the plant. This could be accomplished by manipulating conjugate hydrolysis, transport and/or de novo synthesis. Before this can become of practical value how- ever, more research on IAA dynamics in germinating seeds is needed. l6 DEUTERIUM EFFECTS IN BIOLOGICAL SYSTEMS Deuterium is an isotope of hydrogen containing two neu- trons. The existence of deuterium oxide (heavy water) was first shown by Urey in, 1932. After an initial burst of ex- periments upon its discovery, the number of experiments us- ing deuterium in biological studies decreased. A decrease in price made possible by new separation technologies led to a reawakening of the field which proliferated in the, 1960's. Besides its larger size there are several other impor- tant physical characteristics of deuterium oxide with re- spect to biological systems. The viscosity of Ego is 31% and 18% greater than that of HQO at 50 and 38°C respec- tively. The rate of diffusion is therefore slower in Of) than in HQO. The ionization constant is approximately one- fifth that of HQO, altering the pH of systems in DyD. Long and Bigeilsen (1959) have shown that the rates of acid-base catalyzed reactions may be quite different in H20 as com- pared to 020. At the biochemical level, deuterium exhibits primary isotope, secondary isotope and solvent effects. Primary isotope effects are those due to the difference in reactiv- ity between deuterium and hydrogen bonds to carbon, nitro- gen, oxygen, etc. Bonds to deuterium are more stable and thus require a greater activation energy to break them. In partially deuterated systems, deuterium bonded to nitrogen 17 and oxygen will rapidly exchange with the hydrogen of water. Deuterium bound to carbon does not exchange freely. These deuterium however can be exchanged as a result of keto-enol tautomerism. Secondary isotope effects are those which oc- cur due to deuterium located in positions other than the re- action site. Solvent effects are those due to the physical differences between 020 and H20. In actuality it can be very difficult to distinguish one effect from the others in biochemical reactions. The strength of intermolecular hydrogen and hydrophobic bonds is greater in 020 than H20 (Kresheck eral., 1965) . Proteins dissolved in DTO exhibit a more stable helical structure as a result of altered H-bonding (Tomita axd., 1962.) In contrast non-exchangeable deuterium in amino acid side chains decreases protein stability (Hattori awd., 1964). The alteration of the equilibrium between random coil and ordered helix forms of DNA has been suggested as a primary effect of deuterium oxide (Henderson and Dinning, 1962). A major problem in the use of labeled precursors for metabolic studies is compartmentalization. Applied precur- sors may fail to enter the pool of interest. This problem can be avoided by using a compound, such as deuterium oxide, that distributes itself relatively uniformly throughout the organism. Plants do have some selective capabilities for the uptake of H20 vs 020. Lemna grown on partially deuter- 18 ated medium was able to keep the deuterium content inside the plant 10 to 15% less than that of the media over a two year period (Cope a1d., 1965). The effects of deuterium on growth is dependent to a large extent on enzymatic events. Although no single reac- tion is significantly altered at lOW'EQO concentrations, the sum of all reactions probably results in a metabolic imbal- ance capable of producing severe functional disturbances. Metabolism may also be greatly effected as a result of de- creased penetrability of substrates to specific pools. In the presence of 95-100% [no the overall reaction rate of succinic dehydrogenase was inhibited by about 35%. In 15% or 30% EQO the inhibition was only 5% (Thomson and Klipfel, 1960). Deuterated compounds are not metabolized as rapidly as their non-deuterated counterparts (Erlenmyer awu., 1936, Frei and Aebi, 1958) Based on all of the research done on deuterated enzyme systems it appears evident that (1) deuterated substrates and coenzymes are more tightly bound than are the corresponding normal compounds and (2) the presence of deuterated reactant does not influence the bind- ing of non-deuterated reactant (Thomson, 1963) The effects of deuterium oxide on plants appears to be that of a nonspecific chaotropic agent (Table 3). Deuterium oxide effects every level of plant organization. Crane awd. (1964) and Blake mid. (1964) have studied the effects 19 Table 3. Deuterium effects reported in plants. 20 0302:0080 ONO 22000002 000032: 5? 000.0205 >020: 2F 6000202 003 0:5 5:02.50» 10—03002 0:0 .8008... 05 02,030: ONO 03.3 5 205.5% 2:8 0.200% =< 5.60m .0055 .3525 8—0 0:0 282.: 0:0 2.0509020 :00 .6232: See: 05 0>on0 00230 35:25 .8232: 02:0 05 :05 00320. E03000 293:0: 2:. .009 03w E 0000—: :2? 538m :9... 308 w:=:00m 598 SN 60:20.6: race: b0> 0:03 3:03 ONO $8 5 202:8 05 .Lo .58 a; 50:2 08.: ON: on a 500080 to 5050.5 szm 0 :8 E0000 EEO: :00: 00230 Can Ron 5 3:2: 02:82.50: each... 4:250:05 20 :08... 203 8000 0:0 :08 203 3E2. 0:0 E0: 0030: 02:00:00 .802 602300.. 08809 8: :6 0:0 :05 0003 08.: 600209: 0003 000033 E00250 own 008 0:0 .53 5 63820055. 0:0 “0:08.50 0.03 538» 0ND .82. E 232250: omen? 2005:5092: mats: 00:99: :308 we 0000:2800: 0:0 5:00—08: 228:0 5 00.0200: 0 0:0 .020: 53005302 3:0Ewm0m .m_0>0_ can :2: 3:03 223 :0 28:00: 038200 0:0 0:530: .020 5 00:00:00 002002 03838 33822 .022 0032 5.025 OM 00:9: .20: 5300500.. 5 8000009 0.0000. msot0> 82 woo— $3 $9 3.3 $3 00502 0:0 52:55 Coumotom 0cm 59m .3 :0 302.5 .3 00 0:05 .3 :0 0:23: .3 00 05:02—00:25 338m 0200:» 333% :00» .553. .0 2.0:. 21 of 020 on Menthapipen’ta. Cuttings were grown in nutrient solu- tions containing up to 100% EQO. At the morphological level they found that above 20% Duo there was a proportional re- duction in growth with increasing concentrations. The roots and shoots appeared normal up to 50% Ego except for a reduc— tion in length. No inflorescence primordia were seen at any [no concentration. Histologically the volume of the cells was increased resulting in greatly thickened leaves. The number of vascular elements was decreased and the amount of collenchyma increased in order to provide greater support. Cellulose production was normal, however, the amount of cal- cium-pectate produced was greatly reduced. All of the ef- fects were more pronounced in differentiating tissue. Par- tially deuterated atropine was produced in Atropa belladonna grown in 30% and 50% [no but in smaller amounts than the controls (Uphaus awd., 1963). There appears to be a high degree of selectivity in sensitivity of seeds to 030. Large seeds germinate more readily than small ones, likely as a result of the greater hydrogen content of the food reserves (Blake a1d., 1968). This inhibition may be due to a repression of hydration in [no impairing GA-independent a-amylase synthesis resulting in altered germination as seen in barley (Bhandarkar, 1970). Among all of the seeds tested for germination and grown in tho, rye has shown the least deleterious effects (Siegel a al., 1964) . Stein et a1. (1963) working with Pisium and Zea 22 found that submersion of roots in 80% Ego led to an eventual reduction in the growth rate to zero. The time required to reach the zero rate, as well as the recovery time in HyD, varied with the plant type. Monocots appeared to be more sensitive than dicots and differed in their response to 013. High levels of’EQO have been shown to increase the per- meability of beet roots (Waber, 1984). This effect was the- orized to be due to an alteration in metabolic processes re- quired for membrane maintenance and not to direct action by tho. This was based on the fact that the use of specific protein synthesis inhibitors resulted in a similar response. The rate of protein turnover in Lem/2a fronds was in- creased 50% irntho due to an inhibition of protein synthesis and an increase in protein degradation (Cooke awd., 1979). The increase in protein degradation was found to be the re- sult of an influx of vacuolar proteolytic enzymes due to al- tered tonoplast properties. The use of EQO in studying metabolism offers several advantages over other labels. (1) [no evenly distributes itself throughout the cell and organism. (2) £30 is a more efficient labeller of compounds. (3) [no can easily be fed to plants My00 and does not require any manipulations which might result in artefacts. The use of low E90 concentra- tions does result in a alteration of metabolism. This al- teration however is of a non-specific nature resulting only in a lag of growth. Therefore, Ego would be an ideal label 23 to use for determining if a system makes IAA. The rates of incorporation can be used to determine the relative impor- tance of demmo biosynthesis versus conjugate hydrolysis dur- ing seedling development. 24 INDOLE-B-ACETIC ACID BIOSYNTHESIS IN DEVELOPING SEEDS The site of auxin production in the developing cereal seedling was originally thought to be the coleoptile (for review see Went and Thimann, 1937). This conclusion was based on the fact that auxin would diffuse out of excised coleoptile tips into agar blocks, but not out of the stump (Skoog, 1937). Additionally, removal of the tip stopped growth but growth would resume after regeneration of a "physiological tip" (Dolk, 1930). Today we believe the shoot is the site of hydrolysis of esters transported from the seed (Sheldrake, 1973, Cohen and Bandurski, 1982). Sev- enty years ago, however, there was no knowledge of IAA con- jugates or even auxin "precursors" and the coleoptile tip was widely accepted as the site of auxin biosynthesis. The presence of growth hormone in Awma endosperm was originally shown by Cholodny (1935b), Laibach and Meyer (1935 ), and Pohl (1935). Cholodny obtained growth curva- ture by applying blocks of endosperm moistened with water to the side of the coleoptile. When moistened with alcohol there was no curvature, most likely the result of enzyme in- activation. Pohl depleted seeds of hormone and observed a decrease in growth which led him to conclude that growth hormone is not produced in the coleoptile but is transported up from the seed and activated by the tip. Went and Thimann believed this hypothesis was incorrect. They believed that some precursor, not growth hormone itself, is transported 25 from the seed to the coleoptile where it is converted to auxin. Our current level of knowledge shows that both Pohl and Went and Thimann were correct in some respects. Growth hormones are transported to the shoot from the seed as Pohl concluded, but in an inactive conjugated form (Nowacki and Bandurski, 1980, Komoszynski and Bandurski, 1986, and Chisnell and Bandurski, 1988). The apparently logical con- clusion by Went and Thimann that the transported compound was a precursor has lead to some confusion. The "precursor" which we now know to be IAA conjugates are synthesized FROM indole-3-acetic acid, not the other way around. In the course of conducting a morphological study on the development of the rye seed Nutman (1939) made several conclusions about the production of growth hormones. Corre- lations were found between antipodal and nucellar pillar de- generation and growth of the aluerone and embryo sac. The embryo stops growth when the antipodals have disintegrated, and resume growth when the aleurone layer is initiated (concomitantly with nucellar pillar disintegration). Based on these observations Nutman concluded that growth substance production in the seed is associated with the degeneration of certain tissues. This hypothesis was later supported by Luckwill (1948) who believed IAA was produced via TRP re- leased from proteins upon autolysis. Several other re- searchers have proposed the seed/ovary as the site of hor- mone production. Muir (1942) working with tobacco found 26 more growth substance in the ovary than can be explained by the contribution from the pollen and pollen tube. This was contrary to the views of Gustafson (1939) who felt growth substances from the pollen were responsible for ovary devel- opment. Based on his results, Muir concluded, "growth sub- stances found in the style and ovary are not transferred from the pollen but are produced Dumm as the result of enzy- matic hydrolysis of proteins." In addition it was hypothe- sized that growth hormones move out of the ovary and play a role in the development of the conducting elements in the pedicel and other portions of the ovary/inflorescence. In support of this was the work by Soding (1926) who found that removal of the flower buds greatly decreases growth of the flower stalks. Soding (1926) and Uyldert (1928) suggested that the growth acceleration is due to growth promoting hor- mones produced within the flower bud and young fruit. This suggestion was latter reaffirmed by Soding (1932), Snow (1932) and Kostytschew (1931). Laibach and Meyer (1935) found hormone levels highest during flowering, fruit setting, and germination and lowest during vegetative growth and flower formation of Helianthius. Fertilization was shown to be required to obtain a large in- crease in auxin in the egg and young fruit by Soding (1936), Dollfus (1936) and Judkins (1941). Gustafson (1939) found that while auxin content increased rapidly in seeded fruit, it decreased in seedless fruit. Katunsky (1936a,b) working 27 with Papaver and Crepsis found auxin maximas associated with the development of the nucellus and rapid cell division after synapsis. It was concluded that developing ovules were the centers of hormone production. In doing now classical strawberry experiments, Nitsch (1950) concluded that the achenes supply the developing receptacle with auxin. It was well known by this time that auxins were capable of inducing parthenocarpy. Britten (1950) observed that parthenocarpic kernels most often occurred adjacent to fertilized ones. To explain the development of parthenocarpic maize kernels it was concluded that large amounts of stimulating agents were produced by the fertilized kernels and diffusion of these to adjacent unfertilized embryos induced parthenocarpy. In coniferous seeds it is not known if hormone are pro- duced in situ or translocated into the seed as it develops (Sandberg and Ernsten, 1987). For angiosperms it seems that the old work and theories proposing the seed as a site of auxin production have largely been either forgotten, ig- nored, or both. There has been virtually no auxin biosyn- thetic research conducted with reproductive tissues. Based on the high concentrations of hormones in seeds (relatively speaking), developing seeds or seed tissues should prove to be a valuable system for studying IAA biosynthesis. 28 AUXIN DYNAMICS DURING SEED DEVELOPMENT The levels of IAA during seed development have been de- termined in Zea mays (Avery and etal., 1942, Teas and Newton, 1951, Jensen and Bandurski, 1991), rye (Hatcher and Gregory, 1941 Hatcher, 1943 Hatcher, 1945), wheat (Wheeler, 1972), apple (Luckwill, 1948, 1953), strawberry (Nitsch, 1954), peach (Miller awd., 1987) and bean (Bialek and Cohen, 1989). All seeds examined so far, except for beans, generally have an auxin peak at some developmental stage followed by a rapid decrease as the seed matures. Hatcher and Gregory (1941, 1943, 1945) working with rye and using bioassays were the first to study, and have perhaps given the best account of, the physiology of IAA accumulation in developing seeds . Their first paper showed that no hormone was produced until 14 days after anthesis at which time it underwent a rapid and large increase. Most of the hormone was found in the endosperm. After reaching a maximum, hormone levels rapidly decreased, concurrent with the desiccation of the seed, to a concentration approximately 12% that of the maximum. Avery awd. (1941) discovered that large amounts of IAA could be formed upon alkaline hydrolysis of water extracts of Zea mays kernels from what they called an auxin "precursor". Using alkaline hydrolysis they conducted a comparative study on the auxin content in developing Zea mays kernels of varying heterogenic vigor (1942). While the total auxin content of the dry seeds from the different lines did not vary signifi- 29 cantly the maximum attained during development varied by as much as 50%. They found no absolute relationship between the amount of precursor and free auxin, but free auxin never amounted to more than 12% of the total. Prematurely har- vested kernels lost less auxin upon drying compared to those allowed to dry on the plant. They found that 50% to 70% of the decrease in auxin concentration during maturation could not be explained by destruction during drying. This raises the possibility of some physiological reason for the loss, which to this day remains unknown. They proposed the "precursor" might be a simple ester. However, both methyl and ethyl esters of IAA give a growth response in bioassays while the precursor substance did not. Another possibility was that of an acid amide, possibly of IAA. Armed with the then new technique of alkaline hydroly- sis Hatcher completed a new study of auxins in rye and found 20 times more auxin than found with plain water extraction (1943, 1945). Unlike the findings of Avery awd. (1942) for maize, in rye no precursor substance was formed until after the auxin concentration had reached its maximum. The levels of the precursor continued to increase even in detached seeds suggestiong it is synthesized in the seed. The ratio of the alkali to water extracted auxin remained relatively constant. As the water soluble auxin declined so did the alkali soluble auxin. This decrease was shown not to be due to translocation but occurred.Msflu. In order to account for 30 the stability of the ratio it was hypothesized that: (a) an equilibrium exists between precursor and free auxin during development or (b) the two substances are produced indepen- dently at different rates. Seventy percent of the auxin was found in the aleurone layer in the vicinity of the embryo which itself was found to be lacking auxin. Based on these results it was concluded that the "precursor" is a deriva- tive of already synthesized auxin, and auxin inactivation occurs.Msflu. .Additionally they concluded auxin is not translocated from the mother plant but is produced in the seed from the cytoplasm of disintegrating cells during development. Teas atd. (1951) used a colorometric method to deter- mine IAA levels in developing Zea mays seeds. The general shape of the curve they obtained is very similar to that ob- tained by Avery wad. Unfortunately both groups failed to sample up to the time of maximum fresh weight, and most likely missed the true maximum in IAA content which occurs just before the onset of desiccation (Jensen and Bandurski, 1991). During apple seed development two hormone peaks were observed by Luckwill (1948). The first peak corresponded with the formation of the endosperm and suggests the en- dosperm as the site of hormone production. The second peak occurred after the embryo had finished growth. Luckwill concluded that the hormone plays some part in embryo devel- 31 opment and that high hormone levels and seed numbers are in- volved in preventing premature fruit drop. Very similar re- sults have been obtained in developing peach seeds by sev- eral other researchers (Stehly and Thompson, 1959, Powell and Pratt, 1966, Miller «ah, 1987). The auxin level dynamics of developing bean seeds how- ever, is different from those discussed previously. In all of the developing seeds discussed so far the auxin levels undergo a rapid increase in both free and bound auxin to a maximum and then decrease rapidly as the seed matures. The timing of these stages varies with the type of seed. In beans, the level of total IAA remains stable throughout the developmental period. During development free and ester IAA gradually decrease to a level approximately three times less than that present initially, while amide IAA gradually in- creases to a value five times the initial. Recent work sug- gests IAA stored in bean seeds may play only a minor role in supplying IAA to the developing seedling. Instead IAA re- quired by the seedling for growth is produced denovo (Bialek and Cohen, 1991). Since the mid fifties little research has been done on the auxin levels in developing seeds. This decline in re- search on auxins during seed development may be the result of a lack of any proven function for IAA during seed devel- opment or during the breaking of dormancy. On top of this, 32 other hormones have been identified which definitely func- tion during these times and have proven easier to study. Other areas of IAA research have not been so stagnant. Our knowledge of auxin function, metabolism in other tis- sues, and under various stimuli has since then increased dramatically. The true nature of the "precursor" auxin found by Avery aaL (1941) has since been determined. Large amounts of IAA are bound to various sugars and amino acids. IAA exogenously applied to plants tissues was found to be converted to IAA-aspartate (Andreae and Good, 1955) and to IAA-glucose (Zenk, 1961). The identity of many other conjugates of IAA has also been determined (for reviews see Cohen and Bandurski, 1982, Bandurski dab, 1987). This conjugation of IAA is important in storage, transport, hormonal homeostasis, and protection from peroxidative attack. Developing kernels of Zeamays have proven to be a valu- able tissue for studying the enzymatic conversion of IAA to its various conjugates (Leznicki and Bandurski, 1988, Kowalczyk and Bandurski, 1990, 1991, Kesy and Bandurski, 1990). The similarity in auxin level dynamics between dif- ferent seeds during development is noteworthy. Avery nah (1942) believe the decrease during maturation is due to some physiological reason and is not an artefact of desiccation. The decrease in auxin during maturation has not been studied 33 in detail and such a study may lead to valuable new informa- tion on IAA regulation and function. The in situ production and relatively high auxin levels make developing seeds an attractive system for studying auxin biosynthesis and its regulation. 34 INDOLE-B-ACETIC ACID METABOLISM IN TISSUE CULTURES The advent of tissue culture techniques has brought about the ability to manipulate the genetic makeup of cells in an attempt to modify the plant in a beneficial manner. A major obstacle in this endeavor is the inability to regener- ate whole plants, from not just transformed cells, but cells in general. This is especially true of most major crops in- cluding wheat, maize, barley, oats and soybean. Removal of this obstacle will most likely come through a better under- standing of hormonal regulation of differentiation. IAA most undoubtedly plays a major role in this respect. Most tissue culture systems require an external auxin source, usually 2,4-dichlorophenoxy acetic acid (2,4-D) or napthalene acetic acid (NAA), for sustained growth to occur. IAA is not as effective as its analogs in this respect. Ex- ternally applied IAA is rapidly taken up and metabolized by cells in culture. The rate of inactivation is much slower in autonomous (non auxin requiring) cultures (Rekoslavskaya, 1975). Synthetic IAA analogs are inactivated at a slower rate (Andrae and Good, 1957). These analogs however do not simply serve as a replacement for endogenous IAA. It has been proposed (Lee, 1972, Negutiv awd., 1979) that the role of 2,4-D is to alter the levels of endogenous auxins. It has been shown that normal (auxin requiring) cultures pro- duce IAA at levels commensurate with that of whole plant tissues, and the levels change with the different stages of 35 the culture cycle (Maloney eta1., 1983) . Michalczuk eral. (in press) have reported exogenously applied 2,4-D causes a sig- nificant change in endogenous IAA levels in carrot cell cul- tures. The 2,4-0 somehow confers upon the cells the ability to undergo somatic embryogenesis when transferred to hormone free media. The most striking aspect of IAA levels in tissue cul- tures is the variability seen not only between different species, but even between cultures of the same species (Tables 4 & 5). This makes it difficult to draw many gen- eral conclusions and suggests a greater diversity in IAA metabolism than previously believed. Conclusions can be drawn within a culture type, but care must be taken when making conclusions between different species. Each species in culture (or whole plant) likely has its own particular level of free IAA and maintains that level in a way unique to that culture (or plant). Undoubtedly there exist some general trends, but data from only a small number of cul- tures (and plants) has been collected. Free IAA is consid- ered to be the form of physiological importance, but of equal importance is its ratio to the conjugated forms. The regulation of these two are most certainly intertwined. Currently only one paper has appeared presenting in depth data on the relationship between free and conjugate IAA in a tissue culture system (Michalczuk awfl., 1992). Table 4. IAA levels reported in culture 36 tissue IAA species type (ng/g fwt) year author Soybean T 62-11 1988 Owen et a1. H 12 N 8-20 Carrot N 31' 1979 Sung -2,4-D 128* 5MTR 352* -2,4-D 5145* Pennisetum purpureum N 8 1 9 8 7 Raj aseka ran 01w Sunflower T 45 1979 Chirek N 31 Tobacco T 100 N 35 Sugar Beet N 1400 1981 Keevers wad H 1250 Discorea deltoidea N 1 4 9 1 9 8 1 Kutacek et al. Helianthus annus T 1 0 - 1 3 1 9 8 0 Atsumi N@ 0.2-4 Tobacco T 100 1988 vanOnckelon T 2 0 et al. N <5 Soybean N 2-13 1988 Wyndaele awu N 2-30 H+Kn 7.5-15 H 24-31 T 2-16 T 2 N= normal (auxin added), N@= mutanagized cells, T= Agrobacterium infected, H= habituated, H+Kn= habituated + kinetin, 5MTR= S-methyltryptophan resistant * value approximated from ng/g dry wt assuming 90% moisture 37 Table 5. Maximum IAA levels reported during culture cycle tissue free IAA species type (ng/g fwt) year author Mcotz'ana rustica T 1 7 1 9 8 2 Mousdale N 5 Daucus carom T 9 0 N 6 Helianthus annus T 2 3 N 3 khhmdwe T 6 daigremomiana N 2 Parthenocissus T 2 tricuspidata Daucus carom EN 4 0+ 1992 Michalczuk 6 7 2 t 8! al. N EN 4 0+ 6 0 3 t Panax ginseng N 1 2 0 0 1 9 7 6 Bekker et a1. Polyscias filicifolia N 1 5 0 0 Acer pseudoplantanus N 2 7 * 1 9 8 2 Mol oney & Elliot Acer pseudoplamanus N 4 0 1 9 8 1 Moloney et al. continued on next page Table 5. (cont.) 38 tissue free IAA species type (ng/g fwt) year author Soybean T 3.5 1985 Wyndaele N 3.5 Tobacco N 190 1986 Russian Tobacco T 800 1981 Nakajima Tobacco N 6++ 1987 Koves & H 4++ Szabo Tobacco T 110 1987 El Bahr T 130 eral. N 75 Tobacco T 125 1984 El Bahr N 70 eta]. N= normal (auxin added), T= Agrobacterium infected, H= habituated, 5MTR= S-methyltryptophan resistnat, EN= normal embryogenic, NEN= normal nonembryogenic, t= total IAA * value approximated from ng/g dry wt assuming 90% moisture + estimated from graph ++ estimated based on 20 g of tissue/flask 39 In most cases the level of IAA (free) in tissue cul- tures rises to a maximum prior to the period of rapid mass increase and then declines through the exponential growth phase to a low level as the cultures enter the stationary phase. This holds true for both normal and autonomous cul- tures. The peak in IAA level appears to be associated with the initiation of cellular division and suggests some type of involvement in this regard. This is supported by the fact that the peak in IAA occurs before the mitotic index reaches its maximum (Nishinari & Yamaki, 1979). In addition it has been shown (Gamborg, 1982) that IAA is required during the growth phases of the cell cycle of tobacco cells in culture. The large decrease in IAA coincides with the period of rapid increase in mass. There are of course exceptions to this trend. A number of researchers have reported on cul- tures where the maximum level of IAA occurs during the late exponential or stationary phase (Atsumi & Hayashi, 1978, El Bahr awd., 1984, El Bahr a1d., 1987). These cultures how- ever were of a tumorous type and it is not known how this may affect IAA relations with respect to the culture cycle. There has also been a report of random variations in IAA levels with no correlation to the cell cycle (Wyndaele awd., 1985). The most studied aspect of IAA in tissue culture has been the role of IAA levels in the autonomous growth of 40 crown gall infected and habituated tissue cultures. The T- DNA inserted into the host's genome from the Ti plasmid of Agrobacterium tumefaciens encodes for proteins responsible for the production of IAA from tryptophan via Indole acetamide (IAM) (Kosuge etal., 1966, Inze etal., 1984, Schroeder etal., 1984, Thomashaw awd., 1984). The presence of this alternate path- way diminishes the usefulness of these cultures in studying IAA biosynthesis as it occurs normally in plants. Habitu- ated cells appear as a result of stably inheritable changes in the gene expression which occur at a frequency several orders of magnitude greater than that of normal mutations (Meins, 1981). In general tumorous tissues produce more IAA than non- tumorous ones (Tables 4 & 5) which apparently enables them to initiate the phase of rapid growth. This has been pro- posed, and is generally accepted, as the reason for auxin independence (Beiderbeck, 1977). In a comparison of IAA levels from a number of different species in normal versus infected tissues with respect to time, Mousdale (1982) ob- served that only the normal cultures failed to exhibit a rapid increase in IAA content. It was suggested that the rapid growth phase is triggered by an increase in endogenous IAA above a critical threshold level. In addition to auxin overproduction, auxin independence may be the result of an alteration in sensitivity to endogenous auxin, or the cells could no longer be subject to auxin control of rate-limiting 41 steps in cell growth and division (Mousdale, 1982). An al- tered sensitivity may explain the results of Keever and. (1981) and Kutacek and. (1981) who both observed little dif- ference between normal and habituated cultures. A number of researchers have examined the relationship between tryptophan and IAA levels in tumorous and in non-tu- morous tissue cultures. Most of the data indicate no rela- tionship between tryptophan and IAA content (Atsumi, 1980, Kutacek a1d., 1981, Maloney and Elliot, 1982, El Bahr awd., 1984). This agrees with the findings of Allen and Baker (1980) who found no relationship in the leaves of Rknms communis. In Discorea deltoidea cultures, auxin-habituated cultures possessed a higher anthranilate synthase activity than did normal cultures (Kutacek a1d., 1981). The habituated cell line was also more resistant to L-tryptophan feedback inhi- bition, yet the level of IAA, which was high, was similar to that of the normal cell line. This led them to assume that the tryptophan and IAA pools are relatively independent. Widholm (1972) selected a number of 5-methyl-tryptophan re- sistant cultures for experimentation. Ten of the cultures accumulated high levels of tryptophan but only five were auxin-autotrophic. Therefore tryptophan accumulation alone is not sufficient for auxin-autotrophy. IAA levels in these cultures have not yet been measured. Alternatively, Sung (1979) examined an autonomous S-methyl-tryptophan resistant carrot culture and found it contained high levels of trypto- 42 phan and also contained high levels of IAA. This finding is supportive of a relationship between tryptophan, IAA, and autonomous growth. Maloney and Elliot (1982) measured the levels of tryptophan and IAA in Ann cell culture and found that IAA reached its maximum level well before tryptophan did. This evidence goes against the suggestion by Sheldrake (1973) that IAA levels are regulated by the availability of tryptophan. Additionally no relationship was found between cell autolysis (as a source of tryptophan) and IAA levels. El Bahr and. (1987) examined the activities of two enzymes of the indole pyruvate (IPyA) pathway for IAA in tumorous and non-tumorous tobacco cultures. They found no relation- ship between the activities of tryptophan amino transferase (TAT), Tryptophan dehydrogenase (TDH) and IAA levels. They concluded the increase in IAA seen in the tumorous line was not the result of increased biosynthesis via the IPyA path- way. Black and Hamilton (1976) observed conversion of ”fl:- tryptophan into IAA in soybean callus tissues. They also found that cell free medium could convert.l4C-tryptophan to IAA. Since exogenously applied tryptophan has been shown to enter a pool apparently separate from that of endogenous tryptophan (Sung, 1975) they concluded that either excess tryptophan leaked out of the cell and into the medium and was converted to IAA or the tryptophan might have entered a pool were conversion can occur. 43 Maize tissue cultures as model systems Maize is an ideal plant for studying in tissue culture systems. The biochemistry and genetics of maize has been very well characterized and many genetically well defined lines exist. Cultures initiated from endosperm during the period of high meristematic activity (9-12 DAP) have been used for a number of physiological and biochemical studies. Maize endosperm in culture appears to retain a number of characteristics of the intact tissue. Maize endosperm cul- tures exhibit autonomous growth on media containing mineral salts, sucrose, asparagine, and thiamine (Shannon, 1982). Endosperm cultures of Ricinus communis also require no exogenous hormones for autonomous growth. Cultures of endosperm have the ability to synthesize and store starch, but not to the extent of the intact kernel (Chu and Shannon, 1975). Zeins are also produced (Shimamoto, 1983). This is interesting since at the age from which the cultures were initiated, synthesis of zein begins has yet to begin (Randolph, 1936). In endosperm cultures zein is being produced in actively proliferating tissue, while in intact kernels zein deposi- tion begins at the time when division is nearly complete. Anthocyanins are also produced in cultures initiated from anthocyanin containing endosperms (Straus, 1960, Racchi, 1985). At the ultrastructural level, several differences from intact kernels are apparent (Felker, 1988). In addition to 44 normal storage cells there are meristematic cells and an abundance of highly vacuolate cells. Many cells have an abundant SER, a feature not associated with intact maize en- dosperm. It could not be concluded whether the differences were associated with an altered state induced by the culture technique or represents an amplification of normally minor cellular activities. Miernyk (1987) assayed the medium from maize endosperm cultures and found an abundance of acid hy- drolases. Enzyme activities were similar when compared to those from intact endosperm except for a substantial in- crease in B-galactosidase activity in the cultures. The abundance of SER and the data of Miernyk supports the find- ings of Wink (1984) who suggested the medium serves as an extracellular lytic compartment in tissue culture systems. Maize kernels can be grown DIWUO resulting in viable seeds (Gegenbach, 1977). h1Wno grown kernels offer a unique opportunity to regulate the nutrient supply to the develop- ing kernels for research purposes. Shimamoto and Nelson (1981) examined thiamine transport and found uptake very limited compared to that of sucrose. Since thiamine is a necessary component in most culture media the difficulty in taking up supplemental nutrients may explain the difficulty 35S-methionine, in obtaining auxotrophic mutants. Using Cully and. (1984) compared protein synthesis in developing kernels grown firmno to those grown in the field. They found the DIWUO grown kernels to be comparable to that of the 45 field grown kernels thereby demonstrating the appropriate- ness of the system for future study. Singletary and Below (1989,, 1990) and Singletary awn. (1990) have used develop- ing kernels to study the affects of different levels of car- bon and nitrogen on the production of storage products and associated enzymes. Maize kernels grown DIWUD do not require hormones in the media. Therefore it is reasonable to conclude the ker- nels have the capacity to synthesize the hormones they need. The production of cytokinins by DIWUD grown kernels has been demonstrated (Reinicke and Rubenstein, 19918). In light of the large amounts of IAA produced in developing maize ker- nels (Jensen and Bandurski, 19918), hIWUo kernel culture should be an excellent system for studying IAA biosynthesis. Tissue culture systems are a valuable tool in studying plant hormones. The production of each of the major hor- mones has been demonstrated in a variety of culture systems (Nickel, 1958, Mackenzie and Street, 1970, LaRue and Gamborg, 1971, Nakajima awd., 1981, Wyndaele, 1985, Rajasekasan awd., 1987, Koves and Szabo, 1987). The spe- cific activities of enzymes extracted from tissue cultures can be significantly greater than those from differentiated tissues (Gamborg, 1966). A major difficulty in studying plant hormones has been their presence in small amounts. Those cell lines which exhibit high hormone levels should be particularly valuable in this regard. In culture systems 46 the biosynthesis of the hormones can easily be demonstrated and quantified. Thus, in metabolic studies, the amount and rate of production from labeled precursors can be calculated and the relative significance of that conversion determined. For the study of IAA, those cultures which are autotrophic, yet non tumorous, should prove to be the least complicated and most valuable to study. 47 INDOLE-3-ACETIC ACID BIOSYNTHESIS RESEARCH: WHERE IS IT BEADED? Since the discovery and identification of indole-3- acetic acid (IAA) as a major plant hormone many scientists have conducted experiments in an attempt to elucidate the biosynthetic pathway. Thimann (1935) first suggested tryp- tophan (TRP) as a precursor of IAA based on feeding experi- ments with the fungus Rhizopus suinus. Wildman, Ferri, and Bonner (1948) were the first to present evidence for the conversion of TRP to IAA in higher plants. At various times over the past 50-60 years reports have occasionally appeared suggesting TRP is not a precursor of IAA in higher plants. Working with Awma coleoptile tips Winter (1966) and Thimann and Growchowska (1968) failed to observe elongation when supplied with TRP. On this basis they concluded TRP was not a precursor. Another possible reason for a lack of conversion is that this tissue does not normally synthesize IAA. Black and Hamilton (1971) obtained low rates of conversion of MC-TRP to 1"’C-IAA in Avena coleop- tiles. They believe their low rate of conversion was due to the fact that exogenously applied TRP does not fully equili- brate with the free TRP pool important in IAA biosynthesis. More recently the role of TRP as a precursor has been seriously questioned. Working with a corn mutant containing a defective TRP synthetase Wright awn. (1991) have demon- strated conclusively that TRP is not a precursor of IAA in 48 this plant. When seedlings were grown on media supplemented with (lsN-indole)-TRP there was no incorporation of the 15N into IAA even though IAA levels in the mutant were 10 times that of the control. This indicates there may be at least two biosynthetic pathways, one having a branch point some- where before TRP. Multiple pathways for IAA biosynthesis have been proposed before (Wightman, 1968), but they have always included TRP as a precursor. This coupled with the continued inability to produce unequivocal data substantiat- ing any of the proposed TRP to IAA pathways has slowed ad- vancement in all areas of IAA research compared to that of the other plant hormones. The purpose here is not to criti- cize the volumes of work done over the years but to high- light the steps which need to be taken to unequivocally elu- cidate the IAA biosynthetic pathway(s). Proposed biosynthetic pathways Several pathways from TRP to IAA have been proposed and studied. A diagram of the most widely accepted pathways is shown in Figure 1. The most evidence and the greatest sup- port is for the indole pyruvic acid (IPyA) to indole ac- etaldehyde (IAld) to IAA pathway. A number of putative en- zymes of the IAA biosynthetic pathway have been partially purified but they generally are of a relatively unspecific nature (Wightman, 1968, Muir, 1968, Kutacek, 1985). IPyA is 49 Figure 1. Proposed pathways for indole-3-acetic acid biosyn- thesis in plants 3e. glut; 0000000.: 05.333000 06203303 :33... nun-.83 200:0; 0000001 > s 33 E .0183... 0 000000.001 033380 a 0:383 3.83.5. 51 produced in plants as a result of deamination of TRP by a multispecific aromatic amino acid transaminase (Gamborg, 1965). Transaminases that have been demonstrated in plants have a specificity for TRP significantly lower than that of phenylalanine and tyrosine (Truelson, 1972, Forest and Wightman, 1972, 1974). Additional evidence has come from the promotion of catalysis by adding pyridoxal phosphate and a-keto glutarate, both typical promotors of transamination (Wightman, 1968, Muir and Lantican, 1968). The presence of IPyA has been shown by isolating the 2,4-dinitrophenylhydra- zone derivative of this highly unstable compound (Wightman and Cohen, 1968, Gibson et. a1, 1972,). Positive mass spec- tral identification of IPyA in plants has been made by Cooney and Nonhebel (1989). A TRP dehydrogenase has been found which can convert TRP to IPyA (Ebeid a1d., 1985). The back reaction was found to be more active and it was pro- posed that this would be a possible way to regulate the lev- els of IPyA and IAA biosynthesis. No enzyme has yet been found in plants which can con- vert IPyA to IAAld. An IPyA decarboxylase has been demon- strated in yeast (Sukanya awd., 1971). Due to its highly unstable nature IPyA can spontaneously break down to form IAA. Circumstantial evidence for enzymatic decarboxylation comes from the stimulatory effects of thiamine pyrophosphate and lipoic acid (Gordon, 1961) known cofactors of oxidative 52 decarboxylations. Gibson nah (1972) has also presented qualitative evidence for the decarboxylation of IPyA. The conversion of IAAld to IAA has been demonstrated by a dehydrogenase (Wightman and Cohen, 1968) and an oxidase (Rajagopal, 1971). Both enzymes were found to be mainly cy- toplasmic however they had different pH optima. Kutacek (1985) suggest IAAld oxidase is important in regulating IAA levels. The possible role of tryptamine (TNHZ) as an IAA pre- cursor was first shown by Skoog (1937) who observed coleop- tile curvature with TNH2 after a lag period. The existence of TNH; has been demonstrated in some plants but has not been found in a number of common plants (Schneider dab, 1972). Enzymes which decarboxylate TRP to form TNH¢ have been found in a number of plants (Phelps and Sequira, 1968, Sherwin, 1970, and Gibson nah, 1972a,b). The next step in this pathway is deamination to from IAAld. A number of amine oxidases have been examined in plants (Mann, 1954, Yamazaki nah, 1970). The indole acetamide (IAM) pathway has been demon- strated in Pseudomonas savastanoi (Kosuge er al. , 1966) and Agrobaco teriumatmefaciens (Thomashaw «al., 1984, Inze «al., 1984) The Ti-plasmid of A. tumefaciens contains genes which code for en- zymes that convert TRP to 1AM and IAM to IAA (Thomashaw a 40, 1984, Inze «at, 1984). The plasmid is inserted into the hosts genome during infection. This results in an 53 increase in IAA levels and subsequent tumor formation. Kutacek awd. (1987) have demonstrated both the IAM and IPyA pathways to be operative in infected tobacco cells in culture. Modifications to the pathways in Figure 1 are indicated in Figure 2. The pool of TRP in plants is large compared to that of IAA and this presents a regulatory problem. How can the small amount of TRP needed for IAA biosynthesis be regu- lated? To get around this it has been proposed that D-TRP is a more immediate precursor than L-TRP (Law, 1987, McQueen-Mason and Hamilton, 1989, Tsurusaki awd., 1990). L- TRP is first racemized to the D form resulting in a greatly reduced pool size. D-TRP is then further metabolized via IPyA to IAA. The presence of N-malonyl-D-TRP in plants is well established (Good and Andreae, 1957, Aenk and Scherf, 1963) and may serve as a storage form of TRP (Rekoslavskaya, 1986) as well as a detoxification product (Rosa and Neisch, 1968). N-malonyl-D-TRP accumulates in water stressed plants and decreases upon removal of stress which is accompanied by a large increase in IAA levels (Gamborg awd., 1991). Indole ethanol (IEt) has been identified as a normal constituent of plants (Rayle and Purves, 1967, Sandberg, 1984) and may served as a storage form of precursor. IEt also reversibly forms glycosidic conjugates which may serve as further stor- age forms (Magnus, 1973) 54 Figure 2. IAA biosynthetic pathway variations and regulation mechanisms. 56 Finally Figure 3 shows other pathways which have been studied or proposed. The role of indole acetaldoxime (IAOX) and glucobrassicin as an IAA precursor has been shown in various members of the Brassica family (Kutacek and Kefeli, 1968), and cereals (Bentley and Housley, 1952, Rajagopal and Larsen, 1972, Helming awd., 1987) but is not believed to be widespread in the plant kingdom. steps toward definitive answers A major problem in the study of IAA is its presence in small amounts, generally 1-100 ng/g fwt. When working with such small amounts it is difficult to distinguish between artifact and actuality. It has been demonstrated that radi- olabelled TRP can be converted to IAA with a 30% yield merely by drying (Epstein awfl., 1980). Another compounding aspect to this problem is the amount of labelled and/or un- labeled potential precursors which has been fed to the con- version system being examined. Care must be taken to insure that only physiological levels are used. In their work with Lemna, Baldi et al. (1991) obtained stable isotope labelled IAA from intact plants grown on agar containing 15N-L-TRP. The amount of L-TRP added in order to obtain conversion however was 400 to 1600 times greater than its pool size. They were unable to obtain conversion with D-TRP at any concentration. Even though they did obtain conversion of TRP to IAA, this conversion was determined to be physiologically not 57 Figure 3. Proposed alternate pathways for indole-3-acetic acid biosynthesis. 59 important under normal conditions. These results led them to conclude TRP is not a precursor of IAA in this system. Most of the feeding experiments conducted to date used large amounts of labelled and/or unlabeled potential precursors. The conversion rates obtained with l4C-TRP in most experi- ments are below 0.05%. There is no doubt that these experi- ments have demonstrated the conversion of TRP to IAA. The weakness of these studies is the lack of quantitative evi- dence as to the significance of the conversion. As a re- sult, definitive proof of TRP as an important precursor has not been demonstrated. This shows the need to know the pool sizes of the precursors and products such that the relative conversion of the precursors can be determined and checked for significance. Catabolism and the formation of IAA conjugates are im- portant processes in the regulation of IAA levels (Cohen and Bandurski, 1983). Free IAA is the form active in growth but the conjugates represent the bulk of the IAA in plants (Schulze and Bandurski (1977). Black and Hamilton (1976) and the recent work in Cohen's lab are to date the only ones to examine booth free and conjugate IAA in their systems. Free IAA is readily conjugated and by not examining the to- tal IAA it is difficult to determine the significance of any observed conversion. There has been rapid development in separation tech- nologies over the past 15 years. The majority of the data 60 collected on IAA biosynthesis was completed prior to the re- cent upsurge in new technology. Identification of putative intermediates and IAA has been based largely on paper chro- matography Rfs and response to colorometric tests. Data of this nature does not represent conclusive proof as to the identity of a compound. The presence of interfering or con- taminating compounds cannot be excluded. Definitive proof can be obtained by purifying to a constant specific activity and demonstrating that the specific activity remains con- stant when subjected to additional purification procedures. Positive identification of the molecule then needs to be made by MS, NMR, or IR analysis. The latter two are imprac- tical due to the large amounts required for analysis. Con- clusive evidence can also be obtained with the use of stable isotopes. Proof that one compound has been converted to an- other is easily obtained by demonstrating the presence of the label in the product upon mass spectral analysis. This method is the most reliable, unfortunately the cost and availability of labelled compounds can be prohibitive. Many different tissues have been used to study IAA biosynthesis. Often missing is the evidence that the tissue being used is one that normally produces IAA. The coleop- tile of cereals has been a popular experimental tissue. There is however data suggesting that in some cereals the seedling does not biosynthesize IAA but is totally reliant on the seed stores of the hormone (Pohl, 1935, Sheldrake, 61 1973, Jensen and Bandurski, 1990). IAA is generally be- lieved to be synthesized in the meristematic regions of plants (for reviews see Sembdner, 1981, Sheldrake, 1973). Definitive proof that a system is producing IAA adds strong support to the results and conclusions arrived at using that system. Proof that a system is producing IAA can be ob- tained by measuring IAA levels over time (free and conju- gate) and checking for an accumulation. Additionally the incorporation of stable isotopes from the feeding of general labels such as D20 or 13C-glucose can be used to demonstrate that IAA biosynthesis is occurring. Greater emphasis needs to be placed on how biosynthesis is occurring bump. The use of extracts or tissue sections can lead to misconceptions about what occurs in an intact plant. Cells contain a variety of different compartments and as a result there are enzymes and substrates that nor- mally do not come in contact with one another. Disruption of the cells may result in two materials coming together re- sulting in a reaction not normally seen. Several re- searchers have demonstrated the conversion of TRP to IAA when incubated with catechol at alkaline pH or in the pres- ence of phenolase at lower pH (Gordon and Paleg, 1961, Whitmore and Zahner, 1961). Both researchers doubted the importance of this reaction bnmm but warned of its possible importance when working with plant extracts. The use of m iamlexperiments is still an important tool for studying the 62 IAA biosynthetic pathway. Initial hanoresults however need to be followed up with carefully set up 0‘00 experi- ments using intact plants. Wounding initiates a wide range of responses in tissues including a variety of free radical catalyzed reactions. In experiments using cuttings care must be taken to distinguish between normal and wounding related pathways. The likeli- hood of two possible pathways is an important consideration at this point. One pathway, which normally accounts for only a minor amount of the total biosynthesis may play a more important part in wounded or infected tissues. This can be misleading when trying to determine the significance of a particular pathway. The difficult task for the future is to distinguish between major, minor and artifactual pathways. Plants can be colonized by many species of bacteria (Libbert, 1966). These bacteria can produce IAA (Libbert, 1966, and Wichner and Libbert, 1968) and non-sterile plants were found to contain more IAA than sterile ones (Libbert a ah, 1968). It has been suggested that the production of IAA observed in many earlier experiments was the result of contamination. This series of experiments by Libbert's lab have shown the importance of insuring that any experimental system used be checked for and maintained under sterile conditions. 63 Since it's discovery, many different aspects of IAA bi- ology have been studied. Several basic aspects of IAA metabolism however remain unresolved. There is clearly much we do not know about IAA. There is a high potential for pratical applications of IAA related research. Recent pa- pers indicate a definitive biosynthetic pathway for IAA has not been demonstrated. Even though IAA is widely believed to be synthesized in meristematic zones there is little con- clusive data to back this up. The timing, regulation of production, and IAA levels in various tissues throughout plant development is poorly understood at best. Resolution of these questions will enhance our ability to regulate plant growth. 64 LITERATURE CITED Allen, J. R. F. and D. A. Baker. 1980. 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EXPERIMENTAL I INCORPORATION OF DEUTERIUM INTO INDOLE-3-ACETIC ACID AND TRYPTOPHAN IN ZEdbbUG SEEDLINGS GROWN ON 30% DEUTERIUM OXIDE. 84 85 ABSTRACT The onset of demno synthesis of IAA and tryptophan in seedlings of Zea mays sweet corn, var. Silver Queen was exam- ined. Deuterium labeled water as a general precursor was used to minimize assumptions as to the biosynthetic route. Protium in positions 2,4,5,6, & 7 of the indole ring are non-exchangeable (Magnus amt, 1980). IAA and tryptophan synthesized via the shikimic acid pathway would contain deu- terium in one or more of these positions. Protium on the indene nitrogen, the carboxyl, the amino group (TRP only), or the protium alpha to the carboxyl (IAA only), exchange readily and were removed by base catalyzed exchange prior to GC-MS analysis. We have demonstrated, by means of gas chro- matography-selected ion monitoring mass spectrometry, that 7 day old, dark grown seedlings grown on 30% [no incorporate deuterium into tryptophan, but not into IAA. Randomization of deuterium in the indole ring during mass spectrometry makes difficult to localize the deuterium to the tryptophan ring. Deuterium NMR is used for the determination of deu- terium incorporation in tryptophan. Data will be presented on the localization of the deuterium in the ring structure. 86 INTRODUCTION The endosperm of corn kernels contain large amounts of bound indole-3-acetic acid (IAA) (Haagen-Smit awm, 1942, Avery «1%, 1944, Ueda and Bandurski, 1969, Piskornik, 1975) which is used by the seedling during germination. At what time does the seedling stop relying on these reserves and begin demmo biosynthesis of IAA? The determination of this time is important in the study of the kinetics of IAA levels and transport in seedlings. While the seedling is relying on its IAA reserves, the metabolism of IAA may be considered as a closed system. In a closed system the only inputs of IAA that need be considered are conjugate hydrolysis and transport, allowing for a less complicated picture of IAA metabolism in seedlings. An open system with respect to IAA is difficult to study owing to the lack of a clear demon- stration of a definitive biosynthetic pathway and data re- garding synthetic rate. The onset of demmo biosynthesis of IAA and tryptophan can be determined by growing seedlings on deuterium enriched water and monitoring the incorporation of deuterium into the molecules. The use of deuterium oxide (E50) as a tracer re- moves many of the problems associated with uptake of applied precursors such as compartmentation, specific changes in metabolism (for review see Thomson, 1963) and wounding re- sponses. During demmo biosynthesis involving the formation of the indole rings of IAA and tryptophan, there would be 87 the opportunity for deuterium incorporation through keto- enol tautaumerization. Proton exchange of partially puri- fied samples insures any deuterium present are of enzymatic origin. The presence of heavy isotope abundancies greater than that naturally occurring indicates demmm biosynthesis of the compound has occurred. Tryptophan was chosen to be analyzed because of its structural similarities to IAA and possible common biosynthetic pathway. In this paper we pre- sent data that as of 7 days of age there is demno biosynthe- sis of tryptophan but not of IAA. MATERIALS and METHODS Plant Material Corn kernels Zhamqm cv. Silver Queen (W. Altee Burpee Co.) were surface sterilized for 10 minutes in a 0.5% NaOCl (1:10 dilution of commercial bleach) solution. The kernels were then rinsed a minimum of four times with sterilized distilled water. All materials were sterilized prior to use. Approximately 25 kernels were placed into each of two beakers containing 20 ml of HgO or 30% DyD. The beakers were covered with aluminum foil and placed on a shaker for 24 hours. After this time the kernels were rolled in paper towels, seven kernels per roll, and placed into 500 m1 grad- uated cylinders, three rolls per cylinder. Fifty milliliters of H20 or 30% [ho was added to the graduated cylinders which were then plugged with a cotton ball to 88 maintain sterility and allow gas exchange. All kernel ma- nipulations were carried out in a laminar flow hood with the use of sterile gloves. The kernels were allowed to grow for seven days in the dark at 25°C and 80% relative humidity. After seven days the seedlings were harvested, shoot lengths taken, and then either used immediately or frozen at -20°C for later analysis. Plants grown on 30% EQO show a growth inhibition of 23%. The plants appeared normal except for a slower rate of development. The fresh weight to dry weight ratios were similar in H20 and 30% D20 grown plants (data not shown). Purification of indole-3-acetic acid and tryptophan. Approximately 2.5 gm of shoots were ground in suffi- cient acetone to give a final concentration of 70%. At this time approximately 50,000 cpm of [5-3H] IAA and 50,000 cpm of [5-3H] tryptophan were added to the extract to aid in peak identification. The mixture was allowed to set for one hour at room temperature, after which time the extract was filtered, the residue being saved for a dry weight determi- nation. The filtrate was dried under vacuum at 50°C until nearly dry then redissolved in approximately 2 ml of 50% ethanol. The sample was then loaded onto a DEAE Sephadex column (acetate form, 3 ml bed volume, Pharmacia) and washed with 50 ml of 50% ethanol. The tryptophan is not retained by the DEAE, washing off in the first 10 ml of solvent di- rectly onto a Dowex 50 column (H+ form, 100 mesh, 10 ml bed 89 volume,). The IAA is eluted from the DEAE with a 0 to 2.5% (v/v) linear gradient of acetic acid in 50 % ethanol. The IAA fractions were pooled, dried under vacuum and dissolved in solvent appropriate for HPLC. The sample was chro- matographed on a HPLC (LDC/Milton Roy model CM400 multiple solvent delivery system with a model 3000 spectro Monitor variable UV detector set at 280 nm) equipped with a C18 col- umn (Snacmon, 250 X 4.6 mm ID, Val-U-Pak, Regis). The mo- bile phase was 25% aqueous acetonitrile containing 1% acetic acid. The IAA fractions were pooled, dried under vacuum, dissolved in 100 pl methanol, and methylated with 250 pl of an ethereal solution of diazomethane for 5 minutes at 20°C. The methylated sample was dried under a stream of N2, dis- solved in 300 #1 of solvent and chromatographed on a C18 HPLC column using 45% aqueous acetonitrile. The IAA frac- tions were pooled, dried under vacuum, and taken up in a small volume of acetonitrile for subsequent GC-MS analysis. The Dowex column containing the tryptophan was washed with 20 ml of 50% ethanol and the sample was eluted with 2N NHqOH. The tryptophan fractions were pooled, dried under vacuum and dissolved in 1.5 ml of chromatography solvent and chromatographed under low pressure on a silica gel 60 column (15 ml bed volume) using ethyl ac- etate:isopropanol:water:NH40H (20:20:25:1). The tryptophan fractions were pooled, dried under vacuum, and taken up in solvent for HPLC analysis. The sample was chromatographed 90 on a C18 HPLC column using 0.01M sodium acetate buffer (pH 5.1):Acetonitrile (95:5). The tryptophan fractions were pooled, dried under vacuum, then acetylated with acetic an- hydride:pyridine (4:1) at 25°C for five minutes. The reac- tion mix was then dried at 35°C under a stream of N2, dis- solved in 100 pl methanol, and methylated with 250 p1 dia- zomethane. The sample was then dried at 35°C under a stream of N2, and the monoacetyl, methyl ester derivative of tryp- tophan was dissolved in 30% aqueous acetonitrile and chro- matographed on a C18 HPLC column using the identical sol- vent. Sample recoveries varied from 3% to 25 % and averaged 12%. The fractions containing the derivatized tryptophan were pooled, dried under vacuum, and taken up in a small volume of acetonitrile for subsequent GC-MS analysis. Purification of preparative amounts of tryptophan for NMR analysis was done as above except for a few simple modi- fications. The bed volume of the Dowex 50 column was in- creased to 20 ml and washed with an additional 20 ml of 50% ethanol. HPLC purification was done on a Phenomenex Bond- clone C18 column (300 x 7.8 mm, Snacron) at a flow rate of 3 ml/minute. Purified tryptophan samples were dissolved in CD9C12 for lH-NMR analysis or in CH2C12 for 2H-NMR analysis. Spectra were obtained using a Varian VXR 500 MHz NMR spectrometer. 91 Gc-MS analysis Mass Spectral data was generated with a Hewlett-Packard 5890 GC coupled to a Hewlett-Packard 5970 MS. Samples where separated on a DB-17 fused silica capillary column (15 m X 0.25 mm) purchased from J & W Scientific. Analysis of the data were carried out using MS-MSD 59974J software (Hewlett- Packard). The methyl ester of IAA (MeIAA) was chromatographed us- ing a temperature program consisting of a 1 minute hold at 100°C, then 100°-230°C at 30°C/min. MeIAA had a retention time of 5.1 minutes. The helium carrier gas was at 2 ml/min. Each major mass spectral fragment and it's associ- ated isotope peaks through m+5 were analyzed using separate injections and selected ion monitoring. The monoacetyl methyl ester of tryptophan was chro- matographed using a temperature program consisting of a 1 minute hold at 100°C, then 100°-260°C at 30°C/min. The monoacetyl methyl ester of tryptophan had a retention time of 6.5 minutes and the spectral data was collected in a man- ner similar to that of IAA. The relative abundancies for the isotope peaks for each fragment were compared to those of the control to determine the amount of deuterium incorporation. 92 RESULTS and DISCUSSION The mass spectrum of MeIAA consists of a molecular ion at m/z 189, base peak at m/z 130 and additional fragment ions at m/z 103, and 77 (Figure 1a). The mass spectrum of the tryptophan derivative consists of a molecular ion at m/z 260, base peak at m/z 130 and additional fragment ions at m/z 201, 103, and 77 (Figure 1b). The purification schemes for IAA and tryptophan pro- duced samples which gave single peaks upon GC-MS analysis (Figure 2a,b). The lack of additional peaks makes it possi- ble to rule out the possibility of contamination causing ab- normally high abundancies for the isotope peaks. A partial spectrum for tryptophan fromicqo grown seedlings is shown in Figure 3b. A difference in isotope abundancies from the controls (Figure 3a) indicates the presence of deuterium. The isotope abundancies for the tryptophan from D20 grown seedlings are clearly and significantly different from those of the control. The IAA from treated plants is unchanged. Table 1 summarizes the mass spectral analysis of tryp- tophan and IAA isolated from H20 and 30% D20 grown plants. Natural isotope abundancies account for the approximately 10.0% of the molecules from the control plants with an m/z of 131-135. With the IAA there was no difference in the percent of molecules containing heavy isotopes for H20 or 30% DyDgrown seedlings. This indicates there has be no 93 deuterium incorporation into IAA, and hence, no de nova biosynthesis by seven days of age. With tryptophan however, there was a substantial difference in the amount of heavy isotope containing molecules present. In plants grown on 30% D20, 43.8% of the tryptophan molecules contained heavy isotopes compared to 10.2% for those grown on HQO. Correct- ing for natural isotope abundancies, 32.0% of all tryptophan molecules contain at least one heavy isotope as a result of the experimental conditions. We have clearly demonstrated deuterium incorporation into tryptophan by seven days of age. This however does not constitute definitive proof of de nova indole ring synthesis. Preliminary IAA samples were subjected to a base ex- change to remove any deuterium that might be of non-enzy- matic origin. Previous work done in this lab by Magnus amt (1980) has shown that the 2,4,5,6,and 7 positions are stable to base exchange in 14% KOH at 120°C. After no deuterium incorporation into IAA was found in preliminary samples this step was dropped from the purification scheme. Using tryp- tophan synthetically deuterated in the side chain (D3 2,3,3), the base exchange procedure was found to be ineffec- tive at removing the deuterium from the molecule (Table 2). Deuterium in the side chain positions are stable, unlike that in IAA, so the base exchange step was dropped from the tryptophan purification scheme. Definitive proof of de nova biosynthesis is indicated by the presence of deuterium in 94 the indole ring. As a result of the non-exchangability of the side chain protons in tryptophan, the mere presence of incorporation is insufficient to indicate demmm biosynthe- sis. The tryptophan could be synthesized from some pre- formed indole ring with all of the deuteriums in the side chain. Mass spectral data from these samples can not be used to determine the location of the deuteriums in the trypto- phan molecules. Mass spectral analysis of synthetically side chain deuterated tryptophan (D3 2,3,3) revealed that there is a rearrangement of deuteriums upon ionization such that the deuterium ended up in the benzene ring fragment (Table 3). In the plant samples, the presence of deuterium in this fragment could potentially be from the side chain. The use of mass spectrometry can not conclusively demon- strate that.demmo synthesis has occurred. Location of the deuteriums was therefore accomplished through NMR analysis. Excellent resolution of the ring protons of tryptophan was obtained with the 500 MHz NMR spectrometer (Figure 4a) .AzH-NMR spectrum of tryptophan isolated from seven day old seedling grown in the dark on 30% EQO revealed the presence of deuterium in the 2 and 3 positions of the side chain and the 6 position of the indole ring (Figure 4b). Additional ring positions may be labelled but the resolution is such that this may only be hypothesized. In any case there is definitely deuterium present in the indole ring of trypto- 95 phan, therefore we have conclusively shown that tryptophan is synthesized de nova by 7 days of age. Based on the mass spectral and NMR data, we conclude that as of 7 days of age Zea mays cv. Silver Queen seedlings have yet to begin demmo biosynthesis of IAA. At this point in growth, all of the IAA used by the seedling must come from the hydrolysis of IAA conjugates or from a preformed indole ring. The demmo synthesis of tryptophan, however, has begun by day 7, as indicated by the high level of deu- terium incorporation into the molecules. The lack of de nova synthesis for IAA may be the result of the high level of IAA stored in the seeds (Haagen-Smit er al., 1942, Avery etal., 1944, Ueda and Bandurski, 1969, Piskornik, 1975), and small pool size and slow turnover rate. In view of this data, the metabolism of IAA may be viewed as being a closed system in young seedlings. Tryptophan on the other hand has a very large pool, and the turnover rate is very rapid, increasing the likelihood of deuterium incorporation. Most of the amino acids released by storage proteins during germination are transported to the seedling intact (Oaks, 1965). There is evidence however that the aromatic amino acids are syn- thesized denova (Sircar, 1967, Mitra etal., 1976). This is further supported by the high activity of the pentose phos- phate pathway which provides the precursors for aromatic biosynthesis (Higuchi and Shimada, 1967). 96 Based on a turnover rate of 5 pmol/hr (Bandurski, 1989) there is enough IAA present in Zea mays seeds to meet the seedlings needs for upwards of 6 weeks. The transition to autotrophy in Zea mays seedlings with respect to overall car- bon metabolism has been determined by Deleens mud. (1983, 1984). Using C02 depleted ir113c they found seedlings to be totally dependant on seed carbon reserves for the first 7 days. After 7 days, carbon from photosynthesis begins to appear. The seed continues to supply carbon to the seedling for up to 4 weeks. This further supports our findings that the seedling is totally reliant on seed stores of IAA during early seedling growth. 97 Figure 1. Mass spectral fragmentation patterns for methly- ated IAA (A) and methylated and acetylated tryptophan (B) m/e l89 9 -C-O-CH3 + \ / l H m/e I30 -HCN ' + Q—cmcn m/e l03 l-CZHZ We 77 0 @ NH m/z-260 I 9? l —C—OCH3 E? — CHNH C -CH3 -HCN HZCH * ‘ a (If em —» 00 —» [0%] u :- ~’ CH3 rn/z-201 mil-130 98 Wag-c -o—CH3 \ l H / [0] m/Z=77 0 99 Figure 2. GC-MS total ion chromatograms of methylated IAA (A) and methylated and acetylated tryptophan (B) isolated from Zea mays seedlings grown in 30% D20 for 7 days in the dark. 100 ®.m m.w ®.w mam ®.m m.v F 0 . _ rL|p _ p . _ 4. rms H on mM®.m n u on 000; w 0 9 mum.H 0.00mnnnh&"m> 40 UHF 0.5.5 as; w m v m L . . . . . p 0 . 8 i , a n q . n umw® m u r O: H W. wwwm H o . a D.NUWDDKH&"m> #0 UHF 101 Figure 3. Partial mass spectrum showing the quinolinium ion‘ and its associated isotope peaks from control (A) and methylated and acetylated tryptophan (B) iso- lated from Zea mays seedlings grown in 30% D20 for 7 days in the dark. An increase in isotope abundan- cies for the isotope peaks compared to the con- trols is evidence for the incorporation of deuterium. 102 NdMa£U\aan 30¢»z00 [[03 - - .mw... .. «.2 09 ~00 S: 00. 00A max. \. \— o 000 mm. \ muo.m w n _ mm 000.0 m x u . 2: 30.0 .0. and 0 / 000.0 23000 200.330 . eueaso\ooaz +0.00. - - .mwm - - .vwm - - .nm «a; :3 on— mmx. max. vex.— mnA \. _ a s 000... w. .a mm:u.m. w _ ans 0 / 000.0 Table 1. 103 Incorporation of deuterium into indole-3-acetic acid and tryptophan by Zea mays grown on H20 or 30% [no for 7 days in the dark Abundancies of the isotope peaks for the Base Peak (percent of total) Control 30% 020 Tryptophan m/z 130 89.0 i 0.1 56.2 i 1.6 131 10.19 i 0.3 26.1 i 1.6 132 0.59 i 0.07 12.4 i 1.5 133 0.04 i 0.03 3.9 i 0.7 134 0.07 i 0.05 0.9 i 0.2 135 0.06 i 0.07 0.4 i 0.04 136 0.09 i 0.07 0 08 i 0.06 IAA m/z 130 88.8 i 0.1 88.4 - 0.2 131 10.27 i 0.3 10.04 i 0.1 132 0 59 i 0.09 0.90 i 0.35 133 0 12 i 0.12 0.22 i 0.08 134 0 04 i 0.04 0.07 i 0.05 135 O 06 i 0.03 0.21 i 0.09 136 0.15 i 0.09 0.12 i 0.07 104 Table 2. Percent abundancies of the isotope peaks for the MS fragment at m/z 130 for D3 2', 3', 3', Tryptophan before and after base exchange treatment % abundance m/z standard exchanged 130 1.8 1.8 131 4.9 5.1 132 77.1 82.6 133 9.5 9.7 134 4.9 0.7 135 1.8 0.0 Table 3. Percent abundancies of the isotope peaks for the MS fragment at m/z 77 for D3 2', 3', 3', tryptophan showing movement of side chain deuterium into the benzene ring fragment. m/z % abundance 77 23.4 78 42.9 79 29.0 80 4.1 81 0.5 105 Figure 4A.lH-NMR spectrum of methylated and acetylated tryptophan isolated from Zea mays seedlings grown in 30% D20 for 7 days in the dark. 106 eon 0.5 «.5 «K MK vK 0K m.n PerPh—EP-hbhbbbbebb—bub-phhbhhbbbPP-Ph 0 0 107 Figure 4B.2H-NMR spectrum of methylated and acetylated tryptophan isolated from Zea mays seedlings grown in 30% D20 for 7 days in the dark. 108 w w hzm>4 om 109 LITERATURE CITED Avery, B. 8. Jr., J. Berger, and B. Shalucha. 1944. The total extraction of free auxin and auxin precur- sor from plant tissue. Amer. J. Bot. 28:596-607. Bandurski, R. S. 1989. Auxin transport and metabolism: the mechanism of tropic curvature. Giorn. Bot. Ital. 123:321-335. Berger, J. and G. S. Avery Jr. 1944. Isolation of an auxin precursor and an auxin (indoleacetic acid) from maize. Amer. J. Bot. 31:199-203. Deleens, E. and J. Brulfert. 1983. Phosphoenolpyruvate carboxylase capacity and establishment of au- totrophy in maize seedlings. Physiol. Veg. 21(5):827-834. Deleens, E., N. Gregory, and R. Bourdu. 1984. Transition between seed reserves use and photosynthetic supply during development of maize seedlings. Plant Sci. Letters 37:35-39. Haagen-Smit, A. J., W. D. Leech, and W. R. Bergen. 1942. The estimation, isolation, and identification of auxins in plant materials. Amer. J. Bot. 29:500- 506. Higuchi, T. and M. Shimada. 1967. Changes in activity if D-glucose-6-phosphate : NADP and 6-phospho-d- gluconate : NADPoxidoreductases in relation to lignification of bamboo. Plant Cell Physiol. 8:71-????. Magnus, V., R. S. Bandurski, and A. Schulze. 1980. Synthesis of 4,5,6,7 and 2,4,5,6,7 deuterium-la- beled indole-3-acetic acid for use in mass spec- trometric assays. Plant Physiol. 66:775-781. Mitra, R., J. Burton, and J. E. Varner. 1976. Deuterium oxide as a tool for the study of amino acid biosynthesis. Anal. Biochem 70:1-17. Oaks, A. 1965. The regulation of nitrogen loss from maize endosperm. Can. J. Bot. 43:1077-1083. Piskornik. Z. 1975. Distribution of bound auxins in ker- nels of sweet corn (Zea mays L.) Acta Biol. Cracov. XVIII:1-12. 110 Sircar, S. M. 1967. Biochemical changes of rice seed germination and its control mechanism. Trans. Bose Res. Inst. 30:189-198. Thomson, J. F. 1963. Biological Effects of Deuterium. The MacMillan Co., New York. Ueda, M. and R. S. Bandurski. 1969. A quantitative esti- mate of alkali-labile indole-3-acetic acid com- pounds in dormant and germinating maize kernels. Plant Physiol. 44:1175-1181. EXPERIMENTAL II METABOLISM OF INDOL-3-YLACETATE IN MATURING KERNELS OF ZEA MAYS 111 112 ABSTRACT Mature kernels of Zea mays sweet corn contain one per cent of total indole-3-acetic acid (IAA) as free IAA and the remainder as esters with a small percentage as amide linked conjugates (Plant Physiol. 1977. 60:211-213). The origin of IAA in the kernel has never been determined. The ultimate source is the leaves, but it is not known whether IAA, an IAA precursor, or an IAA conjugate is transported from the plant leaf into the developing endosperm of the kernel. In an attempt to distinguish between the above alternatives, GC-MS assays of free IAA and ester IAA were made at zero to 45 days after pollination. Free IAA remains relatively con- stant throughout the entire period at about 0.2 Emol kernel- 1 and there is essentially no ester IAA until day 10. Ester IAA increases from day 10 to day 45 reaching a final value of 500 fmol kernel-1. Additional experiments have lead us to conclude demmo synthesis of IAA is occurring in the en- dosperm. Since free IAA never accumulates we conclude that IAA biosynthesis in the kernel or transport of IAA precur- sors, and not esterification, is the rate limiting step in the accumulation of IAA in the developing kernel. Thus we established the rate at which IAA accumulated in the kernel and this enabled us later to determine whether that rate of synthesis occurred in the kernel. 113 INTRODUCTION Conjugation and storage of plant hormones in developing plant seeds is a general phenomenon (Avery awm, 1942, Hatcher, 1943, Miller awm, 1987, Bialek and Cohen, 1989). The stored hormones are available as sources of hormone for the developing seedling plant (Epstein aim, 1980), are pro- tected against oxidation (Cohen and Bandurski, 1978) may serve as transport forms (Nowacki and Bandurski, 1980), and ultimately serve in mechanisms for hormonal homeostasis (Bandurski awd.1988). Seeds, as rich sources of plant hor- mones, have been used in agriculture in antiquity to promote rooting of cuttings. Definitive chemical studies began with Cholodny (1935) who recognized that the endosperm of cereals was a rich source of growth hormone after mild treatment that would hydrolyze bound forms. Pohl, Laibach and Meyer, Hatcher and Gregory, Avery awd., [for reviews see Thimann and Went (1937), Cohen and Bandurski (1982)] showed that the seed hormone precursor was readily converted to the same hormone found in the tip of grass coleoptiles. Finally, Heyn (1935) and Skoog (1937) demonstrated that removal of the endosperm made the seedling more responsive to applied IAA thus developing the concept of a seed auxin precursor. In this paper we describe a time course for the biosyn- thesis of free and ester IAA in the endosperm of thmqwu These data demonstrate that free IAA does not build up in the kernel but that the IAA is esterified as rapidly as it 114 reaches, or is synthesized in, the kernel. In much earlier studies, and using less exacting analytical methods, Corcuera (1967), Avery 01m (1942), and Teas and Newton (1951) for maize, and Hatcher, for rye (1943), had described time courses. In an earlier paper, we studied changes in free and ester IAA during maize kernel germination (Ueda and Bandurski, 1969) and observed a similar, but reverse, se- quence of events. Additional data have led us to conclude IAA is produced demmo in the seed (see experimental III). MATERIALS AND METHODS Plant Material Experiments were conducted on sweet corn plants (Zea mays L., cv Silver Queen) grown under field conditions. Ear shoots were bagged prior to silk emergence and then sib pol- linated once the silks had emerged. In an attempt to com- pensate for variability due to weather conditions, two groups of ears were pollinated on different dates. For the IAA determinations two ears from each pollination group were harvested every 5 days from 0 days until 45 days after pol- lination (DAP). Additional ears were harvested at 58 DAP 7 and allowed to dry down at room temperature. Free and total IAA determinations were conducted on fresh tissue. Each sample consisted of kernels from one ear for a total of four samples per age. 115 IAA purification For the determination of free and total IAA, 15 to 90 fresh kernels were extracted with acetone (final conc.= 70%) after being frozen with liquid nitrogen and ground to a fine powder with a mortar and pestle. An internal standard of [13061-1104 (100 pg/sample) and approximately 80,000 cpm of [3H]-IAA for peak detection were added to the samples. The samples were allowed to extract for 18 hours at 4°C then centrifuged at top speed for five minutes in a benchtop cen- trifuge (1300 G, Internationl Equip. Co.) and the super- natant liquid divided for free and total IAA analysis. Free and total IAA were purified using a modification of a method developed by Chen and.(1988). Quantification was accomplished through the use of stable isotope labelled IAA as an internal standard (Magnus, 1980, Cohen awm, 1986). For the determination of free IAA, 6 ml of the ex- tract was diluted with 5 ml of double distilled HQO and ap- plied to a DEAE-Sephadex A-25 column (acetate form, 1.5 ml bed vol., Pharmacia) packed in a 3 ml syringe fitted with a polypropylene frit. Using a vacuum manifold, the column was sequentially washed with 2 ml of 50% ethanol, methanol, ethyl acetate, hexane, ethyl acetate, methanol, 50% ethanol and 50% acetonitrile containing 0.75% acetic acid. Though the packing underwent drastic changes in volume with the use of 100% organic solvents the IAA was still retained. IAA was eluted from the column with 3 ml of 50% acetonitrile 116 containing 2% acetic acid and dried under vacuum at 50 °C. The sample was taken up in solvent and chromatographed on an HPLC (LDC/Milton Roy model CM 4000 pump with a model 3000 spectro Monitor variable UV detector set at 280 nm) equipped with a C18 column (5 micron ODS, 25 X 4.6 cm, Val-U-Pak, Regis). The mobile phase was 20% acetonitrile/water con- taining 1% acetic acid. The 3 ml fraction at the retention time of IAA was checked for radioactivity, and dried.Mluawu The sample was then taken up in 200 pl of methanol and methylated with 200 pl of diazomethane at room temperature for 5 minutes. After methylation the sample was dried at 45°C under a stream of nitrogen, taken up in solvent and rechromatographed on the C18 HPLC column using 45% acetoni- trile/water as the solvent. The IAA fraction was dried m wupa and taken up in acetonitrile for GC-MS analysis. For the determination of total IAA, 2 ml of the extract was dried to aqueous phase Mxmam then an equal volume of 2N NaOH was added to give a final concentration of 1N NaOH. The sample was purged with nitrogen, incubated for one hour at room temperature, acidified to pH 2.5 with 1N HCl, di- luted with 2 ml of double distilled water and applied to a conditioned (washed sequentially with 5 ml of hexane, methanol, water, and 1% acetic acid prior to sample addi- tion) C18 solid phase extraction cartridge (J.T. Baker Co.). The cartridge was washed with 10 ml of double distilled wa- ter and the IAA eluted with 2 ml of acetonitrile. The sam- 117 ple was dried mxmaw, taken up in solvent and HPLC purifica- tion was as above for the free IAA. Final recoveries ranged from 19% to 50% with an averaged of 34%. Gc-MS-SIM analysis GC-MS-SIM analysis of the purified methylated IAA was conducted on a Hewlett-Packard 5890 GC with a capillary di- rect interface to a model 5970 MSD. The GC column was a 15.0 m DB-17 fused silica capillary column (0.25 mm id., 0.25pm film thickness, J & W Scientific). The GC tempera- ture program consisted of an initial 1 minute hold at 100 °C followed by a ramp to 230 °C at 30 OC/minute. The ions at m/z 130, 136, 189 and 195 were monitored and the peak areas measured to determine the 13C/12C ratio. RESULTS Developing kernel of Zea mays contain levels of IAA sev- eral orders of magnitude greater than that reported in vege- tative tissue. For the first 10 days after pollination IAA levels increase only slowly. Beginning 10 DAP (days after pollination) there is over a thousand fold increase in the amount of total IAA reaching a maximum of 90 pg kernel"l at 45 DAP then decreasing by approximately half for dry seed (Figure 1a). The change in IAA levels parallels the in- crease in fresh weight but slightly precedes the onset of dry weight accumulation (Figure 1b). 118 Plotting free and total IAA on a per gram dry weight basis clearly shows IAA levels begin to rise before the on- set of dry weight accumulation and at a rate independent of the change in dry weight (Figure 2a). When the levels of free and total IAA are plotted on a per gram fresh weight basis it is obvious that as rapid kernel growth begins, the concentration of IAA remains relatively constant (Figure 2b). Compared to the amount of total IAA, the free IAA lev- els remain relatively constant at about 1 to 2% of the to- tal. Considered alone the free IAA undergoes a large in- crease between 5 to 20 DAP from 28 ng/kernel to approxi- mately 1000 ng/kernel and then remains at relatively con- stant level for the rest of the growth period (Fig 3). Dur- ing the period of rapid increase in total IAA the ratio of ester to free IAA is maintained at a constant percentage (Figure 4) when computed on a fresh weight, dry weight, or per kernel basis. Free IAA constitutes approximately 1-2 percent of the total the remaining IAA exists as ester conjugates, there was no detectable amide linked IAA (see appendix B). Figure 1. 119 Comparison of changes in the levels of free and total IAA (A) and the accumulation of fresh and dry weight (B) during the growth and maturation of maize kernels. Vertical bars indicate the stan- dard error from the mean of four replicate samples. IAA (pg / kernel) Weight (mg/kernel) 100 90 80 70 60 50 40 30 20 10 600 500 400 300 200 100 120 _ it. Total 13 Free fH—A—A-Ar—A—A—A—A‘A— -——-A l I l l Days After Pollinotion I I lJ / I I 1 l l V F )— - 0 Fresh Wt. B " C) Dry\Nt ‘ ‘. e e "”{D .. Q . .. I 1 L/ / d / f 40 50 60 Dry Seed 121 Figure 2. Comparison of changes in the levels of free and total IAA when calculated on a per gram dry weight basis (A) and a per gram fresh weight (B) basis during the growth and maturation of maize kernels. Vertical bars indicate the standard error from the mean of four replicate samples. 1 000 IAA (lug/g dry wt.) 8 3 8 8 O O O O O _s _a N O 01 O O O O IAA (pg/g fresh wt.) 0' O 122 // A Total IAA- Free IAA A If 0 Dry wt. ' A Total IAA L A Free IAA . 0 Fresh wt. 010 20 30 40 50 60 Days After Pollination Cal-P 010 CO (A O O t. (mg/kernel) 150 123 1 - %%/ 0 b #A— 1 1 1 1 1,1,1 0 10 20 30 40 50 60 D17 860d Free IAA (pg/kernel) Days After Pollination Figure 3. Changes in the levels of free IAA in the kernel during development 100 90 80 70 60 50 40 30 20 10 Ratio of Ester to Free IAA (70) Figure 4. 124 /Z 7/ \e I I I I I I . I/L 7/ 0 10 20 30 40 50 60 00’ Seed Days After Pollination Percent of total IAA in the ester form during ker- nel development. The remaining IAA is in the free acid form. 125 DISCUSSION The accumulation of IAA in the developing kernel can be divided into three phases. These phases are characterized by the periods of: (1) vascular development of the cob, (2) kernel filling, and (3) dormancy induction. During the first 10 DAP, IAA is present in the kernel in quantities typical of vegetative tissue. Only after the cob has reached a particular stage of development around 10 DAP does IAA become available for storage along with other typical storage compounds. This delay may simply be due to a lack of vascular development preventing the transport of IAA or its precursors into the kernel. Preliminary experiments (see appendix A) have been conducted involving the removal of blocks of kernels from ears 5 DAP resulting in a curva- ture of the cob towards the dekerneled area. When 1% IAA in lanolin is applied to the dekerneled area there is no subse- quent curvature of the cob. This suggests the kernels and IAA play a role in the development of the cob similar to what Nitsch found with the removal of seeds from the achenes of strawberries (1950). Based on this, we believe IAA has some physiological role in the development of the cob. As a consequence of development, IAA is catabolized. At this early stage, it may be the nucellus which provides IAA to the growing cob. After cob growth is nearly complete and 126 the nucellus has disintegrated, it is likely that the en- dosperm is then responsible for the demno biosynthesis of IAA for storage. This has been proposed earlier by Hatcher (1943) who, working with rye, conclude the grain was the site of auxin production based on the fact that prematurely harvested grains continued to accumulate auxin. Additional studies show that on hormone free media, in vitra grown Zea mays kernels produce almost normal levels of IAA and endosperm suspension cultures also produce IAA demno (see experimental III). Beginning with the period of rapid growth, large amounts of ester IAA appear in the kernels. The level of total IAA in the developing kernel clearly increases as the kernel matures. Earlier investigators working with Zea mays, also observed a large increase in IAA during development (Avery awfl., 1942, Teas and Newton, 1951). After reaching maximum levels the total IAA decreased 80% or greater de- pending on the variety or inbred used (Avery arm 1942). Throughout development, sugary endosperms contained more IAA than starchy ones (Avery etal., 1947, Teas etal., 1952) . The developmental stage at which maximum IAA levels were reached also varied with the variety or inbred. The levels of IAA were expressed on a per gram dry weight basis in their stud- ies. The choice of units greatly affects the appearance of the data whether it be per kernel (Figure 1.), per gram fresh (Figure 2b.) or dry (Figure 2a.) weight as has been 127 suggested by Bialeck and Cohen (1989). In these earlier studies they failed to sample to the maximums in fresh weight and likely missed the true IAA maximum. Again this may be the result of how they calculated their IAA levels. When calculated on a per gram dry weight the IAA maximum oc- curs early during development (figure 2a). Differences be- tween the previous work and the data presented here may be due to the use of primitive analytical techniques, the vari- ety of maize, and in the case of Hatcher, the grain studied. In seedlings of Zea mays around 88% of the total IAA ex- ists as ester forms, the remainder as free (Bandurski and Schulze, 1977). In contrast to this, while the kernel is rapidly growing and accumulating IAA, the ester forms ac- count for 97 to 99% of the total. This is indicative of the fact that as free IAA appears in the kernel it is immedi- ately conjugated. This consistency can be explained by ex- amining the thermodynamics of the first two steps of conju- gation ending in the formation of IAA nwo-inositol (IAInos). A G (calories) IAA + UDPG -—————+ 1-O-IAGlu + UDP +1400 1-0-IAGlu + myo-inositol ——————+ IAInos + Glu -4200 IAA + UDPG + myo-inositol ——————+ IAInos + UDP + Glu -2800 For every 1400 calories there is a one order of magnitude shift in the equilibrium of the reaction. A A G of -2800 calories for the sum of these reactions approximates a 99:1 128 ratio of ester to free IAA. This is identical to what we observed in the developing kernel. Therefore the observed rise in free IAA during grain filling is most likely only a result of the kinetics of the conjugation reactions. The difference between the ratio of ester to free IAA between seedlings and the developing kernel is notewOrthy. This sug- gests a different or more simplified control mechanism oper- ates in the kernel. It seems likely that production of IAA is regulated by some as of yet unknown means and it is the availability of free IAA that regulates the levels of IAGlu synthase and other enzymes involved in conjugation. From the present study we can conclude that a control mechanism is operating that prevents accumulation of free IAA in the endosperm of the developing kernel. That such rigid control should be operative during both ripening and germination is indicative of the importance of controlled levels of the hormone both in seed and vegetative growth. Unlike corn, it has been reported by Bialek and Cohen (1989) that during bean seed development the level of total IAA on a per seed basis remains fairly constant while the percentages of free, ester and amide change. A possible ex- planation for the lack of change in the amount of total IAA could be that IAA in bean seeds is produced elsewhere in the plant and then transported to the seed. This is exactly op- posite to what we have observed in corn. In a study on the location of indole alkaloid biosynthesis in hxmwa it was 129 concluded that alkaloids were produced in the leaves and then transported to the seed (Mockaitis awm, 1973). This again is opposite what we have observed in corn for IAA, but may represent an intrinsic difference between monocots and dicots. More recently, Bialek and Cohen have shown that de mun synthesis provides the developing bean seedling with IAA. In this system, IAA conjugates in the seed have little or no role in this regard (1991). This is contrary to what we have found with maize seedlings where there is no de nova biosynthesis and the seedling is totally reliant on IAA from conjugates stored in the seed (see experimental I). No role for IAA in the regulation of embryo growth is known at this time. The changes in total IAA levels run parallel to the change in total protein. A time course of total protein levels in corn during kernel development shows a plateau between 25 and 35 DAP (Ingle arm, 1965). We ob- served an identical plateau at this age in the levels of to- tal IAA. These two may in fact be related developmental processes as was suggested by Bialek and Cohen for beans (1989), but no direct evidence for this relationship has been demonstrated. Several researchers have been using in vitra culture of Zea maw'kernels to examine the biochemical and physiological aspects of kernel growth and development (Cully wwd., 1984, Singletary and Below, 1989, 1990, Singletary ald. 1990). These researchers report that growth of kernels in culture 130 is roughly equivalent to growth of kernels in the field. We propose the use of hIWUokernel culture as a powerful tool for studying the biosynthesis of IAA and it's regulation. We believe this system is ideal for elucidating the complete biosynthetic pathway for IAA. The high levels of IAA in Zaz mam kernels indicate this pathway is extremely active in this tissue, making the system easier to work with. Prelim- inary studies with endosperm suspension cultures also indi- cates their ability to synthesize IAA de nova. We are cur- rently conducting further experiments to determine the fea- sibility of these systems for the study of IAA metabolism. 131 LITERATURE CITED Avery, B. S. Jr., J. Berger, and B. Shalucha. 1941. Auxin storage as related to endosperm type in maize. Bot. Gaz. 103:806-808. Avery, B. 8. Jr., J. Berger, and B. Shalucha. 1942. Auxin content of maize kernels during ontogeny, form plants of varying heterotrophic vigor. Amer. J. Bot. 29:765-772. Bandurski, R. S. and A. Schulze. 1977. concentration of indole-3-acetic acid and its derivatives in plants. Plant Physiol. 60:211-213. Bandurski, R. S., A. Schulze, M. F. Desrosiers, P. J. Jensen, B. Epel, and D. M. Reinecke. (1988). Relationship between stimuli, IAA, and growth. In: Plant Growth Substances 1988. Pharis, R. and S. Rood, eds. Springer-Verlag, Heidelberg. Bialek, K. and J. D. Cohen. 1989 Free and conjugated indole-3-acetic acid in developing bean seeds. Plant Physiol 91:775-779. Bialek, K., L. Michalczuk, and J. D. Cohen. 1991. Auxin metabolism in bean seedlings. Plant Physiol. 96(s):77. Chen, KH., A. N. Miller, G. W. Patterson, and J. D. Cohen. 1988. A rapid and simple procedure for purification of indole-3-acetic acid prior to GC-SIM-MS analysis. Plant Physiol. 86:822-825. Cholodny, N. 1935. Uber das keimungshormon von gramineen. Planta 23:289-312. Cohen, J. D. and R. S. Bandurski. 1978. The bound auxins: Protection of indole-3-acetic acid from peroxidase-catalyzed oxidation. Planta 139:203- 208. Cohen, J. D. and R. S. Bandurski. 1982. Chemistry and physiology of the bound auxins. Ann. Rev. Plant Physiol. 33:403-430. Cohen, J. 0., B. G. Baldi, and J. P. Slovin. 1986. 1306- [Benzene ringJ-indole-3-acetic acid. A new in- ternal standard for quantitative mass spectral analysis of indole-3-acetic acid in plants. Plant Physiol. 80:14-19. 132 Corcuera, L. J. 1967. Estudio de indolacetilinositide en grano de maiz. Thesis. Universidad Catolico de Chile, Santiago, Chile. Cully, D. E., B. G. Gegenbach, J. A. Smith, I. Rubenstein,.J. A. Connelly, and W. D. Parka5 1984. Endosperm protein synthesis and L-[ S] methionine incorporation in maize kernels cul- tured DIWUD. Plant Physiol. 74:389-394. Epstein, E., J. D. Cohen, and R. S. Bandurski. 1980. Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol. 65:415-421. Hatcher, E. S. J. 1943. Auxin production during develop- ment of the grain in cereal. Nature. 151:278- 279. Heyn, A. N. J., 1935. The chemical nature of some growth hormones as determined by the diffusion method. Proc. Kan. Acad. Wetensch. 38:1074-1081. Ingle, J., D. Beitz, and R. H. Hageman. 1965. Changes in composition during development and maturation of maize seeds. Plant Physiol. 40:835-839. Magnus, V., R. S. Bandurski, and A. Schulze. 1980. Synthesis of 4,5,6,7 and 2,4,5,6,7 deuterium-la- beled indole-3-acetic acid for use in mass spec- trometric assays. Plant Physiol. 66:775-781. Miller, A. N., C. S. Walsh, and J. D. Cohen. 1987. mea- surement of indole-3-acetic acid in peach fruits (Prunus persica L. Batsch cv Redhaven) during devel- opment. Plant Physiol. 84:491-494. Mockaitis, J. M., A. Kivilaan, and A. Schulze. 1973. Studies of the loci of indole alkaloid biosyn- thesis and alkaloid translocation in Ipamea violacea plants. Biochem. Physiol. Pflanzen 164:248-257. Nitsch, J. P. 1950. Growth and morphogenesis of the strawberry as related to auxin. Am. J. Bot. 37:211-215. Nowacki, J. and R. S. Bandurski. 1980. Abm-inositol es- ters of indole-3-acetic acid as a seed auxin precursor of Zflzmaw L. Plant Physiol. 65:422- 427. 133 Singletary, G. W. and F. E. Below. 1989. Growth and com- position of maize kernels cultured hififia with varying supplies of carbon and nitrogen. Plant Physiol. 89:341-346. Singletary, G. W. and F. E. Below. 1990. Nitrogen-in- duced changes in growth and metabolism of devel- oping maize kernels grown thno. Plant Physiol. 92:160-167. Singletary, G. W., D. C. Doehlert, C. M. Wilson, M. J. Muhlitch, and F. E. Below. 1990. Response of enzymes and storage proteins of maize endosperm to nitrogen supply. Plant Physiol. 94:858-864. Skoog, F. 1937. A deseeded Awma test method for small amounts of auxin and auxin precursor. J. Gen. Physiol. 20:311-334. Teas, H. J. and A. C. Newton. 1951. Tryptophan, niacin, and indoleacetic acid in several endosperm mu- tants and standard lines of maize. Plant Physiol. 26:494-501. Teas, H. J., J. W. Cameron, and A. C. Newton. 1952. Tryptophan, niacin, indoleacetic acid, and car- bohydrates in developing sugary and starchy maize kernels. Agron. J. 44:434-438. Thimann, K. V. and F. w. Went. 1937. Phytohormones. MacMillan, New York, NY. Ueda, M. and R. S. Bandurski. 1969. A quantitative esti- mate of alkali-labile indole-3-acetic acid com- pounds in dormant and germinating maize kernels. Plant Physiol. 44:1175-1181. EXPERIMENTAL III METABOLISM OF INDOLE-3-ACETIC ACID IN ENDOSPERM SUSPENSION CULTURES OF ZE4AbHG 134 135 ABSTRACT The kernel of Zea mays accumulates large amounts of IAA during development. The origin of this IAA has not yet been determined. A clue to this question is the knowledge that Zea mays endosperm can grow in vitra on media containing no growth hormones. This suggests the endosperm can make its own IAA, and is therefore the site of synthesis in the de- veloping kernel. We have conducted a time course study us- ing endosperm cells in suspension culture to determine if indeed they are synthesizing IAA. Levels of free IAA reached a maximum of 65 ng-gfwt'l on the fourth day after subculture. The total IAA reached a maximum of 620 ngwgfwt' l on the eight day after subculture, 2 days after the maxi- mum in fresh weight. No amide IAA was found. Clear evi- dence that these cells are indeed synthesizing IAA is demon- strated by calculating the IAA levels on a per flask basis. Between the second and eighth days after subculture there was an increase of nearly 10 pg of total IAA in the flask. During this period the increase in fresh weight was only 10 gms. The high levels of IAA being produced by these cells make them an ideal system for studying the biosynthesis of IAA and its regulation. 136 INTRODUCTION The existence of a growth regulating compound in plants was first proposed by Ciesielski in 1871 (Ciesielski, 1872). It was then more than fifty years before the structure of IAA was determined (Kogl 01%, 1934), and shown to be an en- dogenous component of plants in 1942 (Haagen-Smit). A definitive pathway for IAA biosynthesis in plants has yet to be demonstrated. This shortcoming has slowed progress in the other aspects of auxin physiology such as regulation and mode of action. A biosynthetic pathway of IAA involving tryptophan has been well established in several different bacterial species (Kosuge, arm, 1966, Sequira and Williams, 1964, Thomashaw awm, 1984). In plants, tryptophan has also been suggested to be a precursor of IAA. Several lines of evidence including lack of growth induction by tryptophan (Winter, 1966, Thimann and Grochowska, 1968) and questions about microbial contamination (Libbert awm, 1966) contra- dict this hypothesis. More conclusive evidence that dis- proves the role of tryptophan as a precursor in Zea mays seedlings has been presented recently (Wright awd., 1991). It has been proposed that two pathways for the biosynthesis of IAA may exist, a major one involving a pathway excluding tryptophan, and one from tryptophan which may be associated with stress (Bandurski awd., 1992). The literature on the various theoretical precursors and biosynthetic pathways is extensive yet a definitive 137 pathway has not been demonstrated in plants. One of the problems responsible for the slow progress in this field is the lack of a system that is unequivocally producing IAA dc mun and does so at a readily quantifiable rate. Only re- cently working with Lemna (Baldi et al. 1991) and with whole mutant seedlings of Zea mays (Wright etal. 1991) has the pro- duction of IAA from labelled precursors been definitively demonstrated. The endosperm of Zea mays kernels contain large amount of IAA ester conjugates which are used by the seedling during germination (Ueda and Bandurski, 1969). Working with rye in the 1940's Hatcher proposed that the increase in IAA during seed development was not the result of transport from the mother plant but that IAA was synthesized within the seed. Zea mays endosperm suspension cultures also have been initi- ated and grown for many years without the need for hormone supplementation (Shannon 1982). Given the likelihood that IAA is indeed produced de nova by developing Zea mays kernels we undertook a study to determine if endosperm cells of Zea mays produce IAA demno in culture. In this report we will show that IAA is produced de novo and at levels greater than in normal vegetative tissue. 138 MATERIALS AND METHODS Plant Material Suspension cultures of endosperm cells derived from the Zea mays inbred A636 were kindly provided by Dr. Jack Shannon of the Pennsylvania State University. The cells were grown on liquid medium containing Murashige and Skoog salts, 3% sucrose, 2 g/L of asparagine and 0.4 mg/L of thiamine HCl. The pH was adjusted to 5.6 prior to autoclaving. Cultures were grown in the dark at 29°C on a rotary shaker at 120 RPM. Two one liter stock cultures grown for seven days af- ter transfer served as a source of innoculum for the experi- mental flasks. Each stock culture was subcultured into one set of six 250 m1 flasks, each containing 80 ml of media. Every two days after subculture one flask from each set was harvested, fresh weight determined and the tissue lyopholized for later analysis. Zea mays kernels (hybrid B73 X LHSl) grown in vitra were provided by Dr. Fred Below of the University of Illinois. The kernels were grown on solid medium containing Murashige and Skoog salts, 15% sucrose, 2 g/L of asparagine and 0.4 mg/L of thiamine HCl. No hormones were added to the media. Kernels were harvested at 20 and 32 days after pollination. IAA Purification For the determination of free IAA 5% of each sample, based on dry weight, was extracted with 70% acetone under vacuum for 2 hrs. at room temperature. [13C6]-IAA (100 139 pg/sample) as an internal standard and approximately 80,000 dpm of [3H]-IAA for peak detection were added to the sample. After two hrs the sample was centrifuged at top speed for five minutes in a benchtop centrifuge (1300 G, International Equip. Co.) and the supernatant decanted off. The super- natant was then reduced in volume to aqueous phase and then diluted with two ml of water. The sample was then loaded using a vacuum manifold onto a preconditioned 10 mm X 1 cm semi-prep guard column (Upchurch Scientific) loaded with C18 pellicular packing (Whatman). The preconditioning, loading and washing procedures are identical to those of Chen awd. (1988) for a C18 SPE column. The column was conditioned by washing sequentially with 5 ml of hexane, methanol, water, and 1% acetic acid. After washing with 10 ml of water, the precolumn was inserted in place of the injector loop on the HPLC (LDC/Milton Roy model CM 4000 pump with a model 3000 spectro Monitor variable UV detector set at 280 nm) and the sample was eluted off the precolumn onto the analytical C18 column (5 micron ODS, 25 X 4.6 cm, VAL-U-PAK, Regis) with the HPLC solvent (20:80 Acetonitrile:1% Acetic acid). The peak containing the IAA was dried down then taken up in 200 pl of methanol and methylated with 200 pl of diazomethane at room temperature for 5 minutes. After methylation the sam- ple was dried at 45 °C under a stream of nitrogen, taken up in acetonitrile for GC-MS analysis. 140 For the determination of total IAA an additional 5% of the sample, plus the internal standard, was extracted with 1N NaOH under vacuum for two hrs at room temperature. The sample was then acidified to pH 2.5 with 1N HCl and applied to the precolumn and purified as above for the free IAA. Final recoveries for both free and total IAA ranged from 20% to 50% with an averaged recovery of 36%. GC-MB-SIM analysis GC-MS-SIM analysis of the purified methylated IAA was conducted on a Hewlett-Packard 5890 GC with a capillary di- rect interface to a model 5970 MSD. The GC column was a 15.0 m DB-17 fused silica capillary column (J & W Scien- tific: 0.25 mm id., 0.25pm film thickness). The GC temper- ature program consisted of an initial 1 minute hold at 100 °C followed by a ramp to 230 0C at 30 °C/minute. The ions at m/z 130, 136, 189 and 195 were monitored and the peak ar- eas measured to determine the LQQAZC ratio. RESULTS AND DISCUSSION The growth curves (fresh and dry weights) of the Zea mays endosperm suspension cells during the course of the culture cycle are shown in Figure 1. The curves are typical for cells in culture inoculated at a high density. IAA levels determined at two day intervals are shown in Figure 2. IAA biosynthesis by these tissues supports the hypothesis that 141 the endosperm is a site of IAA production in the developing kernel. In proposing a new model for studying IAA biosynthesis it is essential to show that the system is producing IAA de mno under normal conditions. The levels of free IAA during the growth cycle of the endosperm suspension cells are shown in Figure 2a. The maximum in free IAA occurs at four days after subculture. In other cultures where the levels of IAA have been determined the majority also have their free IAA maximum early during the exponential growth phase (Bekker a aL, 1976, Moloney and Elliot, 1982, Mousdale, 1982). It has been proposed that this is important in initiation of the exponential growth phase in autonomous and habituated cul- tures (Beiderbeck, 1977, Mousdale arm, 1985). The high levels of free IAA we observed may explain the autonomous nature of our cultures. Normal cultures require the addi- tion of an auxin to maintain growth. Rather than acting as an auxin itself, 2,4-D has been shown to promote IAA biosyn- thesis in cell cultures (Moloney axm, 1983, Michalczuk a (H., 1992). In normal cultures the cells do not produce enough IAA for autonomous growth, such cultures require ad- dition of an external auxin for growth to occur. The levels of total IAA increase up to a maximum of 12.6 pg/flask eight days after subculture and then decreases to 6 pg/flask. The maximum in total IAA occurs 2 days after the maximum in fresh weight (Figure 2b). The continued in- 142 crease in total IAA after the maximum in fresh weight has been reached is noteworthy. At this time the significance of this is unclear. Presenting the data on a pg/flask basis most clearly shows that Zea mays endosperm suspension cells are indeed producing large amounts of IAA. 9.5 pg of IAA was produced per flask in six days therefore the biosyn- thetic rate is approximately 66 ng/hr/flask. Since only in- organic salts, sucrose, thiamine and asparagine are present in the media, the increase in the levels of IAA is direct proof that demmo synthesis has occurred. Preliminary exper- iments indicate a large amount (up to 20 pg/flask) of IAA is excreted into the media. It is likely therefore that the biosynthetic rate may be up to twice that mentioned above. The curve for IAA levels in the endosperm suspension culture is very similar to that in developing kernels (Jensen and Bandurski 1991). The cultured cells appear to retain the characteristics of the intact kernel with respect to IAA dynamics. The apparent IAA biosynthetic capacity is only 10% that of the intact kernel on a per gram dry weight basis. Another major difference between the intact kernels and the cultured cells is the percent of the IAA in the es- ter form. In the developing kernels the ester IAA is main- tained at around 98% of the total for most of the develop- mental period (see experimental II). In the suspension cells the ester IAA reaches a maximum of only 92% of the to- tal, which is similar to observed levels in vegetative tis- 143 sue. This suggests that while in the kernel IAA is produced for storage, in the suspension cells the IAA produced is ac- tively being utilized by the cells. Moloney and Elliot have reported on IAA levels in nor- maliuxrcell cultures (1982). In their system, free IAA in- creases to a maximum of approximately 275 ng/g dry weight as compared to our system where the maximum level of free IAA is approximately 1000 ng/g dry weight. Unfortunately they did not determine the level of IAA conjugates present in their samples as is true for almost all similar studies in other cultures. the levels of free and total IAA have been measured in carrot suspension cultures (Michalczuk awm, 1992) and are similar to what we have reported. In their embryogenic line however, the levels of IAA drop when 2,4-D is removed from the medium. In the non-embryogenic line only a small decrease was observed when grown on 2,4-D free media. The amount of free and total IAA found in DIWna cul- tured kernels is shown in Table 1. There has clearly been an increase in the amount of IAA present. The kernels were cultured on hormone free media so the observed increase in IAA is clear evidence that the kernel contains all of the enzymes required to biosynthesize IAA. Therefore, the large amounts of IAA appearing in the kernel during development are synthesized in the kernel, likely in the endosperm. 144 We have shown that Zea mays endosperm cells in suspension culture and.hrWnagrown kernels can synthesize IAA. Addi- tionally, we know the rate of production which will allow us to determine the significance of any conversion of applied precursors to IAA. In doing so we can evaluate the impor- tance of a variety of purported precursors of IAA to deter- mine which pathway(s) is operating in these cells. The high capacity of these cells to produce IAA is an- other attractive feature of this system. Any conversion of precursors should be readily observable and not require any rigorous or time consuming analytical procedures. This sys- tem should therefore yield results which are significantly above background with a high proportion of precursors being converted to IAA. Endosperm cells in suspension culture are an ideal sys- tem for studying IAA biosynthesis. In the future we hope to further characterize the IAA metabolism of this system, in- cluding examining the apparent excretion of IAA into the me- dia. Once this has been completed we can begin testing var- ious compounds for their involvement in the production of IAA. 145 25 I j I I I I I N O I _5 (II I Fresh wt. (gm/flask) 3:; 1'2 " 0’0-0—0 t: \o 10 - \ / - g 0.8 - 0 5 t 0.4 i0/ 5 0.0 I I I I I I 0 2 ‘I 6 8 10 12 O I I I I I I I 2 4 6 8 1 0 1 2 Days of Culture Figure 1. Fresh and dry weight of Zea mays endosperm cultures during the culture cycle. Culture were inoculated at a high density to obtain a rapid growth rate. 146 Figure 2. Comparison of changes in the levels of free and total IAA (A) and the accumulation of fresh and dry weight (B) during the life cycle of Zea mays en- dosperm suspension cells. Vertical bars indicate the standard error from the mean. 147 A B I- 0 0 0 0 - mu mu _ _ _ mu m m w. m m. m m m m m m $8005 .3 020 33005 .3 0300 10 Days of Culture 148 Table 2. Synthesis of IAA and IAA esters by DIWND cultured kernels of Zea mays. IAA (pg'kernel'l) Day 20 Day 32 Free 3.5 3.5 Total 16 33 149 LITERATURE CITED Baldi, B. G., B. R. Maher, J. P. Slovin, and J. D. Cohen. 1991. Stable isotope labeling 0‘00, of D- and L-tryptophan pools in Lemna gibba and the low in- corporation of label into indole-3-acetic acid 95:1203-1208. Bandurski, R. S., M. F. Desrosiers, P. J. Jensen, M. Pawlak, and A. Schulze. 1992. Genetics, chem- istry, and biochemical physiology in the study of hormonal homeostasis. Plant Growth Sub- stances, Beiderbeck, R. 1977. Pflanzentumoren. Verlag Eugen Ulmer, Stuttgart. Bekker, A. M., L. S. Gurevich, N. V. Mikhailova, and L. I. Slepyan. 1977. Dynamics of endogenous IAA in auxin-dependent strains of tissue cultures from Panax ginseng and Polysciasfilicifolia. Fiziol . Rast . 24:841-844. Ciesielski, T. 1872. Untersuchungen uber die abwartskrum- mung der wurzel. Beitr. Biol. Pflanz. 1:1-31. Haagen-Smit, A. J., W. D. Leech, and W. R. Bergen. 1942. The estimation, isolation, and identification of auxins in plant materials. Am. J. Bot. 29:500- 506. Jensen, P. J. and R. S. Bandurski. (1991). IAA and IAA ester content of developing kernels of Zea mays. Plant Physiol. 96:(S)28. Kogl, F, A. J. Haagen-Smit, and H. Erxleben. 1934. Uber ein neues Auxin (“Heteroauxin”) aus Harn. XI Mitteilung A. Physiol. Chem. 228:90-103. Kosuge, T., M. G. Heskett, and E. E. Wilson. 1966. Microbial synthesis and degradation of indole-3- acetic acid. I. the conversion of L-tryptophan to indole-3-acetamide by an enzyme system from Pseudamonassavastanoi. J. Biol. Chem. 24l(16):3748- 3744. Libbert, E., S. Wichner, U. Schiewer, H. Risch, and W. Kaiser. 1966. The influence of epiphytic bacte- ria on auxin metabolism. Planta 68:327-334. 150 Michalczuk, L., T. J. Cooke, and J. D. Cohen. 1992. Auxin levels at different stages of carrot so- matic embryogenesis. Phytochem. 31:1097-1103. Moloney, M. M. and M. C. Elliot. 1982. Tryptophan and indole-3-acetic acid accumulation in Adv cell cultures and its relationship with cell autoly- sis. Planta 156:326-331. Moloney, M. M., J. F. Hall, G. M. Robinson, and M. C. Elliot. 1983. Auxin requirements of sycamore cells in suspension culture. Plant Physiol. 71:927-931. Mousdale, D. M. 1982. Endogenous indolul-3-acetic acid and the growth of auxin-dependent and auxin-au- totrophic (crown gall) plant tissue cultures. Biochem. Physiol. Pflanzen. 177:9-17. Mousdale, D. M. A., C. Fidgeon, and G. Wilson. 1985. Auxin content and growth patterns in auxin-de- pendent and auxin-independent plant cell and tissue cultures. Biol. Plant. 27:257-264. Sequeira, L. and P. Williams. 1964. Synthesis of in- dol eacet ic ac id by Pseudomanas solanacearum . Phytopath. 54:1240-1246. Shannon, J. c. 1982. Maize endosperm cultures. In: Maize for Biological Research. W. F. Sheridan, ed. Plant Molecular Biology Assoc. Charlottesville, VA. Thimann, K. V. and M. Grochowska. 1968. The role of tryptophan and tryptamine as IAA precursors. In: Biochemistry and Physiology of Plant Growth Substances. F. Wightman and G. Setterfield, eds. Runge press, Ottawa, Canada. Thomashaw, L. S., S. Reeves, M. F. Thomashaw. 1984. Crown gall oncogenesis: evidence that a Ti DNA fro the AyfimmWMm Ti plasmid encodes an enzyme that catalyzes synthesis of indoleacetic acid. Proc. Nat. Acad. Sci. USA 81:5071-75. Ueda, M. and R. S. Bandurski. 1969. A quantitative esti- mate of alkali-labile indole-3-acetic acid com- pounds in dormant and germinating maize kernels. Plant Physiol. 44:1175-1181. Wichner, S. and E. Libbert. 1968. Interactions between plants and epiphytic bacteria regarding their 151 auxin metabolism I. detection of IAA-producing epiphytic bacteria and their role in long dura- tion experiments on tryptophan metabolism in plant homogenates. Physiol. Plant 21:227-241. Winter, A. 1966. A hypothetical route for the biogenesis of IAA. Planta 71:229-239. Wright, A. D., M. B. Sampson, M. G. Neuffer, L. Michalczuk, J. P. Slovin, and J. D. Cohen. 1991. Indole-3-acetic acid biosynthesis in the mutant maize arangepericarp, a tryptophan auxotroph. Science 254:998-1000. EXPERIMENTAL IV ISOLATED IMMATURE ZEA MAYS ENDOSPERM AS A MODEL SYSTEM FOR STUDYING INDOLE-3-ACETIC ACID METABOLISM 152 153 ABSTRACT The endosperm of Zea mays sweet corn contains as much as 0.5 pmol of esterified indole-3-acetic acid (IAA) in a sin- gle kernel. It was not known where, in the plant, IAA syn- thesis occurred: in the leaves, and then transported to the kernel as IAA or an IAA precursor, or in the kernel from sugars biosynthesized in the leaves. To that end, this lab- oratory has demonstrated that developing kernels cultured in fine on hormone and tryptophan free media continued to accu- mulate large amounts of IAA. Further, endosperm tissue in suspension culture grow freely on hormone free media and re- tain almost 2% of the IAA biosynthetic rate of the attached kernel. Therefore it is certain that the endosperm of.&m mays contains all of the enzymes required for the de nova biosynthesis IAA. With the knowledge of the large capacity for IAA synthesis of the maize endosperm this system would be ideal for examining putative IAA precursors for their ability to sustain IAA synthesis. To determine the feasi- bility of this system we have examined the conversion of l4C-anthranilic acid to IAA in isolated endosperm. An- thranilic acid was used because it is common to all proposed pathways yet occurs early enough so as not to presuppose one pathway over another. Data relating to the rate of IAA syn- thesis from anthranilic acid and proof as to the identity of the IAA will be presented. 154 INTRODUCTION It was has long been assumed that Tryptophan (TRP) is a precursor of Indole-3-acetic acid (IAA). Thimann (1935) was the first to suggest TRP as a precursor of IAA based on his work with the fungus Rhizapus suinus. Wildman et al. (1947) pro- vided the first demonstration of the conversion of TRP to IAA in plants. Numerous studies have been conducted since then which corroborate these findings. Recently several reports have been published which pre- sent evidence that tryptophan is not a precursor of IAA in some plants (Baldi 01d. 1991, Jensen and Bandurski 1990, Wright awu. 1991). The most dramatic of these used a tryp- tophan auxotroph mutant of maize (Wright axd. 1991). Em- 15N-indole)-tryp- bryos were germinated on media containing ( tophan and after 6 or 10 days when the IAA was isolated there was no incorporation of the 15N into the IAA molecules, even though the mutant produced 10 times more IAA than the non-mutant. These experiments demonstrate the need to carefully re- examine the question of the biosynthetic pathway of IAA. Those experiments conducted in the past have failed to un- equivocally demonstrate one pathway or the other in plants. To avoid this problem in the future a number of steps need to be taken in order to provide more conclusive results. It must be demonstrated that the system normally makes IAA. Many studies on IAA biosynthesis have been conducted 155 using cereal coleoptiles. There is evidence however that the coleoptile of some cereals do not make IAA and are to- tally reliant on seed stores of the compound (Pohl 1935, Sheldrake 1973, Jensen and Bandurski 1990). Conversion of precursors to IAA using such systems may be the result of a pathway(s) not normally operative in the intact plant. The pool sizes of the potential precursors and products should be determined so the relative importance of a com- pound as a precursor can be determined. There is a vast amount of data demonstrating the conversion of TRP to IAA (for reviews see Schneider and Wightman, 1974, Marumo, 1986) but the relative importance of this conversion has not been demonstrated. In a recent report Baldi and. (1991) grew lemna on media containing 15N-L-Trp and was able to demon- strate the appearance of 15N in IAA only when the level of Trp applied was 400-1600 times that of the normal pool size. InuNI1SN-D-Trp was fed, no conversion was observed at any feeding level. On the basis of pool size and the amount of labelled IAA produced they concluded Trp was not a signifi- cant precursor of IAA in Lemna. There must be conclusive proof of the identity of any labelled compound produced as a result of metabolism. Iden- tification of reaction products has often been made on the basis of a compound's Rf on paper chromatography. The ra- dioactivity at the Rf's of interest is then determined and conclusions made based on this information. This however 156 does not constitute conclusive proof. When using radioac- tive precursors the product(s) first need to be purified to a constant specific activity to insure the radioactivity is associated with a particular peak. The compound must then be positively identified by mass spectral or some other qualitative analysis. When possible a system that produces large amount of IAA should be used. The low levels of IAA, especially when compared to that of many proposed precursors, increases the difficulty in differentiating between biological and arti- factual conversion. One potential source of error that has been demonstrated is the conversion of radioactive Trp to IAA with a 30% yield by simply drying in a flash evaporator (Epstein 01m, 1980). Using a system with a high capacity for IAA biosynthesis would help guarantee any conversion ob- served was of a biological nature. The endosperm of maize contains large amounts of IAA (Haagen-Smit eral, 1942, Avery etal., 1944, Ueda and Bandurski, 1969, Piskornik, 1975), stored mostly as ester conjugates (Epstein awi, 1980). During kernel development, IAA is produced in the seed and then rapidly conjugated for storage (see experimental II). The endosperm should there- fore contain all of the enzymes required for IAA biosynthe- sis and conjugation. We have found that isolated endosperm system retains its ability to synthesize IAA. We tested the capacity of this system to biosynthesize IAA by using LU}- 157 anthranilic acid as a precursor. Anthranilic acid was used because all IAA biosynthetic pathways proposed to date have this compound in common yet anthranilic acid occurs early enough so as not to presuppose one pathway over another. At this stage we do not wish to make conclusions as to the biosynthetic pathway of IAA. Rather, we concentrated on the suitability of using a maize endosperm system for further IAA biosynthesis research. MATERIALS and METHODS: Ears of thmam'cv. Silver Queen were harvested between 22 and 30 days after pollination and used immediately. The ears were surface sterilized for 10 minutes in 10% bleach and then rinsed 4 times with sterile water. Several en- dosperms were removed from the kernels, placed in pairs in sterile microfuge tubes and gently homogenized with a glass rod. Additional endosperms were frozen at -80°C for later analysis to determine the freezing stability of the system. Alternatively many endosperms were place in a sterilized mortar, homogenized, and then transferred to microfuge tubes in amounts approximately equal to two endosperms. To each tube approximately 250,000 dpm (8.6 nM) of anthranilic acid was added. Control samples were boiled for five minutes and cooled prior to the addition of the radiolabel. The samples were allowed to incubate for 0, 4, 12, and 24 hours, after 158 which the samples were analyzed immediately or frozen with liquid nitrogen and stored at -80°C for later analysis. IAA purification Total IAA was determined using a modification of the method developed by Chen and. (1988). To each sample an equal volume of 2N NaOH was added to hydrolyze the conju- gates and any remaining enzymes. Approximately 80,000 cpm of [3H]-IAA was added to the samples for determining recov- eries and to aid in peak detection . After 1 hour at room temperature the samples were then acidified to pH 2.5 with 1N HCl, and centrifuged for five minutes in a microfuge. The supernatant was applied to a conditioned (column was washed sequentially with 5 ml of hexane, methanol, water, and 1% acetic acid prior to sample addition) C18 solid phase extraction column (J.T. Baker Co.). The column was washed with 10 ml of double distilled water and the IAA eluted with 2 ml of acetonitrile. The sample was dried under vacuum and taken up in solvent for HPLC purification. The sample was further purified on an HPLC (LDC/Milton Roy model CM 4000 pump with a model 3000 spectra Monitor variable UV detector set at 280 nm) equipped with a C18 column (5 micron ODS, 25 X 4.6 cm, Val-U-Pak, Regis). The mobile phase was 20% ace- tonitrile/water containing 1% acetic acid. The 3 ml frac- tion at the retention time of IAA was then checked for ra- 159 dioactivity (Beckman LS 1701). Final recovery values ranged from 32% to 70% with an average of 56%. Some samples were additionally purified as follows. The fraction containing the putative IAA was dried under vacuum then taken up in 200 pl of methanol and methylated with 200 pl of an ethereal solution of diazomethane at room temperature for 5 minutes. After methylation the sample was dried at 45 °c under a stream of nitrogen, taken up in sol- vent and rechromatographed on the C18 HPLC column using 45% acetonitrile/water as the solvent. The radioactivity in the 3 ml fraction at the retention time of IAA was then deter- mined. The sample was then dried under vacuum and taken up in acetonitrile for GC-MS analysis. GC-MS analysis GC-MS-SIM analysis of the purified methylated IAA was conducted on a Hewlett-Packard 5890 GC with a capillary di- rect interface to a model 5970 MSD. The GC column was a. 15.0 m DB-17 fused silica capillary column (J & W Scientific; 0.25 mm id., 0.25pm film thickness). The GC temperature program consisted of an initial 1 minute hold at 100 oC followed by a ramp to 230 °C at 30 °C/minute. The spectrometer was set to scan from m/z 50 to m/z 350. RESULTS The amount of radioactivity associated with the IAA HPLC peak afteI'14C-anthranilic acid feeding for various in- 160 cubation times is shown in Table 1. As much as 42,500 DPM was found to be associated with the IAA peak after 24 hrs. of incubation. Two to eight percent of the total radioac- tivity applied was recovered in the IAA fraction. Based on the original specific activity, approximately 1.2 pg of an- thranilic acid was added per experiment. Using an average conversion percentage of 6%, then roughly 72 ng of labelled IAA has been produced in 24 hours at a rate of 17 pmol/hr. The number of DPM associated with IAA continued to in- crease as incubation times were increased (Fig 1.) leveling off between 12 to 24 hours 3H-IAA was added at the beginning of purification to aid in peak identification and for determination of recover- ies. The ratios of 14C to 3H in the samples at various points of the purification process are shown in Table 2. As expected the ratio decreases as other radioactive metabo- lites are removed from the sample. The ratios after both HPLC steps however remained unchanged, indicating no addi- tional metabolites were removed following methylation and a second HPLC step. A GC-MS analysis of the IAA fraction re- sulted in a single peak (Figure 2a) with a spectrum identi- cal to that of an IAA standard (Figure 2 b,c). 161 Table 1. DPM recovered in IAA fraction after HPLC purifica- tion step. IAA was isp41ated from Zea mays endosperm after incubation with -anthranilic acid. Results of 3 separate feeding experiments. incubation times (hrs) sample 12 24 24 1 17,850 (3.5)* 42,539 (7.9) 29,891 (4.9) 2 11,200 (2.0) 40,997 (7.6) 42,292 (7.6) 3 20,050 (3.5) 28,053 (6.4) ‘ value in parenthesis is percent of applied l4C con- verted to IAA 162 40000 J I I I I ’1_ A ‘i 0 Q) .0.) ° 1 2 30000 ~ ._ . .. La C) 3 '2: 0 20000 - - a. g f ,5 10000 - - 2 a. c: O I I I I I 0 5 10 15 20 25 Incubation time (hrs) Figure 1. Time course showing DPM associated with IAA frac- tion from isolated endosperm incubated with (>- anthranilic acid. 163 Table 2. Ratio of 14C to 3H .in IAA fraction at various stages of purification. sample‘ pre-SPE post-SPE post lst HPLC post 2nd HPLC 1 2.318 0.648 0.089 0.089 2 2.015 0.890 0.100 0.104 3 2.661 0.690 0.119 0.120 .all sangzle were incubated for 4 hrs. with l4C-anthranilic H IAA was added at the beginning of purification. acid. 164 Figure 2. (A) GC-MS chromatogram of HPLC fraction containing methylated IAA pprified from isolated endosperm incubated with CFanthranilic acid. (B) Mass spectrum of single peak observed in GC-MS chro- matogram. (C) Mass spectrum of a methylated IAA standard. 165 0mLM£U\wme D.N¢FVZEH&"¢F¢D #0 UHF SQH Ema SVH QNH 88H 80 U m. 0. n // u OI mm: ems nu . w \\\ mum.N O D.HCDFmHH&"CF¢D #0 nC_E NN®.wv mam CMUW a QWLMLU\wme Emm SWH GVH GNa 88H @m H Ji— . . .J . . m :1. .. £01.. . .._. . L . ...?..... 32%;..." ..o....?...w.._.+ a q \ n / mm: \ w. mm: mm“ R w \ mum . m 0 D.NCFVZCH&"IFIQ #0 nCPE www.mu NHV CMUW e n.C_EV 0E_F w m V m H i p _ r ii p r . . I r L a q n ww®.N u P wMS V W O a 166 DISCUSSION The kernel of Zea mays contains more IAA than any plant tissue examined thus far (Bandurski and Schulze, 1977) The IAA, stored as esters can approach a concentration of 0.5 pmol/kernel (Jensen and Bandurski, 1991). We have previ- ously demonstrated that the IAA is synthesized in the kernel from simple sugars and an amino donor, and is not synthe- sized in the leaves and transported to the kernel as IAA or an IAA precursor (Jensen and Bandurski 1991). The seed as a site of auxin production has been known for many years (Nutman, 1939, Muir, 1942), but has largely been ignored as a tissue to use for IAA biosynthesis experiments. Based on this we believe the endosperm of maize contains all of the enzymes required to synthesize IAA. Additionally the large amount of IAA found in the kernel suggests the endosperm has a high capacity for IAA production. In most experimental systems studied thus far, less than 0.05 percent of the applied radioactive precursors have been converted to IAA. In our system we have observed con- version rates of up to 8 percent. The counts we obtained were significantly greater than the background and that ob- tained with boiled controls (data not shown). Conversions of this magnitude raise little doubt that we are indeed ob- serving IAA biosynthesis in our system. Reported values for IAA turnover range from 1-10 pmol/hr in the shoot and up to 70 pmol/hr in germinating kernels of Zea mays (for review see 167 Bandurski, 1989). We have calculated the rate of IAA biosynthesis in our system to be 17 pmol/hr. This value, which is already greater than most observed values is based purely on the amount of radiolabelled anthranilic acid fed, and does not take into account the endogenous pool of an- thranilic acid. As a result this value is an underrepresen- tation of the actual rate. In the future we will measure the pool size of anthranilic acid and other precursors so we can obtain a accurate rate of IAA biosynthesis. Many of the early studies on IAA biosynthesis did not use rigorous methods to positively identify the metabo- lite(s) of interest. As a result, conclusive proof that the radioactivity associated with that metabolite is actually due to labelled metabolite has often not been presented. By adding 3H-IAA to our samples we have been able to provide strong evidence that the radioactivity we observe is in fact due to labelled IAA produced through the metabolism of an- thranilic acid. We have purified our samples to a constant ratio of'l4C to 3H and therefore conclude the radioactivity' observed was as IAA. It is highly unlikely that another metabolite would co-elute with both the free acid and methy- lated forms of IAA from an HPLC column. Contamination is indicated by changes in the 14C to 3H ratio as purification proceeds. This ratio will continue to change until on all contaminating metabolites have been removed. At this point the rates will remain constant throughout any further pu- 168 rification steps. Definitive proof that the compound we isolated is in fact IAA has been demonstrated through GC-MS analysis. We know that maize kernels biosynthesize IAA in situ, and have shown that isolated endosperm tissue retains this ca- pacity. Large amounts of IAA can be produced by this system and at a rate at least comparable to observed IAA turnover rates. In all likelihood the rate is probably several times greater than reported here. With such high activity the precursor pool sizes will be more readily determined. Pre- liminary experiments indicate this enzyme system is stable to freezing (data not shown). This will insure a plentiful year-round supply of experimental material. We believe this system will prove to be very useful in our further studies on IAA metabolism. 169 LITERATURE CITED Avery, B. S. Jr., J. Berger, and B. Shalucha. 1944. The total extraction of free auxin and auxin precur- sor from plant tissue. Amer. J. Bot. 28:596-607. Baldi, B. G., B. R. Maher, J. P. Slovin, and J. D. Cohen. 1991. Stable isotope labeling MImw, of D- and L-tryptophan pools in Lemna gibba and the low in- corporation of label into indole-3-acetic acid 95:1203-1208. Bandurski, R. S. 1989. Auxin transport and metabolism: the mechanism of tropic curvature. Giorn. Bot. Ital. 123:321-335. Bandurski, R. S. and A. Schulze. 1977. concentration of indole-3-acetic acid and its derivatives in plants. Plant Physiol. 60:211-213. Chen, KH., A. N. Miller, G. W. Patterson, and J. D. Cohen. 1988. A rapid and simple procedure for purification of indole-3-acetic acid prior to GC-SIM-MS analysis. Plant Physiol. 86:822-825. Epstein, E., J. D. Cohen, and R. S. Bandurski. 1980. Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol. 65:415-421. Haagen-Smit, A. J., W. D. Leech, and W. R. Bergen. 1942. The estimation, isolation, and identification of auxins in plant materials. Am. J. Bot. 29:500- 506. Jensen, P. J. and R. S. Bandurski. (1990). Characteriza- tion by NMR of tryptophan isolated from seedlings of Zea mays grown on 30% deuterium ox- ide. Plant Physiol. 93:(S)69. Jensen, P. J. and R. S. Bandurski. (1991). IAA and IAA ester content of developing kernels of Zea mays. Plant Physiol. 96:(S)28. Marumo. S. 1986. Auxins. In: Chemistry of Plant Hormones. N. Takahasi, ed. CRC Press, Inc. Boca Raton, Fl. pp.9-56. Muir, R. M. 1942. Growth hormones as related to the set- ting and development of fruit in Nicotiana (abacum. Amer. J. Bot. 29:716-720. 170 Nutman, P. S. 1939. Studies in vernalisation of cereals VI. The anatomical cytological evidence for the formation of growth-promoting substances in the developing grain of rye. Annals Bot. III(11):731-761. Piskornik. Z. 1975. Distribution of bound auxins in ker- nels of sweet corn (Zea mays L.) Acta Biol. Cracov. XVIII:1-12. Pohl, R. 1935. Uber den endospermwuchstoff und die wuch- stoffproduktion der koleoptilspitze. Planta 24:523-526. Schneider, E. A. and F. Wightman. 1974. Metabolism of auxin in higher plants. Ann. Rev. Plant Physiol. 25:487-513. Sheldrake, A. R. 1973. Do coleoptiles produce auxin? New Phytol. 72:433-447. Thimann, K. V. 1935. On the plant growth hormone pro- duced by Rhizapus suinus.J. Biol. Chem. 109:279-291. Ueda, M. and R. S. Bandurski. 1969. A quantitative esti- mate of alkali-labile indole-3-acetic acid com- pounds in dormant and germinating maize kernels. Plant Physiol. 44:1175-1181. Wildman, S. G., M. G. Ferri, and J. Bonner. 1947. The enzymatic conversion of tryptophan to auxin by spinach leaves. Arch. Biochem 13:131-144. Wright, A. D., M. B. Sampson, M. G. Neuffer, L. Michalczuk, J. P. Slovin, and J. D. Cohen. 1991. Indole-3-acetic acid biosynthesis in the mutant maize orange pericarp, a tryptophan auxotroph. Science 254:998-1000. APPENDEXES 171 172 APPENDIX A Amide IAA Content in Kernels of zanmam'var. Silver Queen. Amide linked IAA conjugates are abundant in beans (Bialek and Cohen, 1989), in contrast to that observed in corn (Epstein arm, 1980). 1N NaOH is sufficient to hydrolyze ester-linked IAA conjugates. Hydrolysis of amide- linked IAA conjugates requires incubation in 7N NaOH at 100°C for one hour. Mature seeds from the same planting previously used for the survey of IAA levels in developing kernel were assayed for amide-linked IAA. These assays were conducted to check the previous findings and determine if amide-linked IAA has a function in the seeds of Zea mays. Materials and methods: Twenty-five dry seeds were frozen with liquid nitrogen and ground to a fine powder with a mortar and pestle. Two aliquots each equivalent to one kernel were weighed out. Samples were hydrolyzed in either 1N or 7N NaOH containing 1 pg of [EX%]-IAA as an internal standard and approximately 50,000 cpm of [3H]-IAA as a radiotracer. The sample in 1N NaOH was hydrolyzed for one hour at room temperature. The sample in 7N NaOH was hydrolyzed for 1 hour at 100°C. Hydrolysis was carried out in Teflon PFA vials (Tuf-tainer, Pierce Chemical Co.). The caps were fitted with an inlet and outlet for nitrogen purging gas. The nitrogen gas was 173 passed through an oxygen scrubber and saturated with water prior to reaching the samples. After hydrolysis, the sample were acidified to pH 2.5 with HCl and centrifuged at 1300 G for 5 minutes. The acidified hydrolysates was purified for GC-MS analysis using SPE mini columns and two HPLC steps as described in experimental II. Results and Conclusions both hydrolysis procedures gave results that were nearly identical. The 1N NaOH hydrolyzed sample resulted in a value of 57,000 ng/kernel of total IAA. The 7N NaOH hydrolyzed sample resulted in a value of 56,700 ng/kernel of total IAA. Based on the lack of a difference in IAA amounts between the two hydrolysis procedures, there is no amide- linked IAA in the kernel of Zea mays var. Silver Queen. 174 APPENDIX B Affect of the Kernel on Cob Development in Zea mays. Studies by Nitsch (1950, 1954) have shown that removal of the seed greatly affects the development of the receptacles of strawberries. This effect is believed to be the result of a loss of growth promoters, presumably IAA, produced in the seed. Asymmetric growth could be obtained by removing the seeds from portions of the receptacle. The cob of an ear of corn is very similar in anatomy to the receptacle of strawberries. An experiment was conducted to determine the role of the kernel, and any IAA produced therein, in cob development. Large amount of IAA appear in the kernels of Zea mays during development (see experimental II). Cob growth is presumably regulated to some degree by IAA. Removal of kernels prior to the period of rapid cob growth might potentially cause a dramatic change in growth. The kernel may be the source of IAA, which in turn controls growth. The kernel may also somehow regulate the levels of IAA appearing in the cob, or may have no affect at all. Materials and Methods At 5 DAP (days after pollination) a block of 72 kernels (6 rows X 12 kernels) was removed from the mid portions of several ears of sweet corn (Illini Xtra Sweet). To accomplish this, the husks were sliced with a razor blade 175 and pulled back to expose the kernels. The kernels were then removed by gently prying with a small spatula. The wounded area was then covered with lanolin, or lanolin containing 1% IAA (w/w). At 25 DAP the ears were harvested and the resultant growth observed. Results and Conclusions Ears to which lanolin alone had been applied to the dekerneled area generally exhibited curvature towards the dekerneled area (Figure 1A). This may be the result of a decrease in IAA on the dekerneled side resulting in Asymmetric growth. Those ears which were treated with lanolin containing 1% IAA generally maintained a straight growth pattern (Figure 1B). This conceivably is the result of the applied IAA supplementing what was lost upon kernel removal. While by no means conclusive, this data suggests in situ biosynthesis is the source of IAA which appears in the kernel during development. 176 Figure 1. (A). Cob development after removal of a 6 X 12 block of kernels from the ears five days after pollination and (B) subsequent application of 1% IAA in lanolin (w/w). N,” Wain»-.. : I 0 (cm) 2 4 6 8 10 12 14 6 Rows REMOVED + 1% IAA IN LANOLIN (w/w) 178 LITERATURE CITED Bialek, K. and J. D. Cohen. 1989 Free and conjugated indole-3-acetic acid in developing bean seeds. Plant Physiol 91:775-779. Epstein, E., J. D. Cohen, and R. S. Bandurski. 1980. Concentration and metabolic turnover of indoles in germinating kernels of Zea mays L. Plant Physiol. 65:415-421. Nitsch, J. P. 1950. Growth and morphogenesis of the strawberry as related to auxin. Am. J. Bot. 37:211-215. Nitsch, J. P. 1955. Free auxins and free tryptophan in the strawberry. Plant Physiol. 30:33-39. HIGH 1 1an STATE UNIV. LIBRQRIES 1|WINVIIINIWW||WWWWI 93007935780 312