I ‘T‘ w Iv T T H! T, W THU T» "Nil H w] ‘T {‘I T ‘ Tvl IN T .I "T “H 'i [T m {if < ‘l T ‘Tl H MT 4 V T MOVEMENT OF lNDOLE~3~ACETTO ACID FROM THE ENDOSPERM TO THE SHOOT OF ZEA MAYS L. Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY PATRICTA LEE HALL 1977 LIBRARY E'migaq State 2% Umvcz'sz'ty g e..- ABSTRACT MOVEMENT OF INDOLE-3-ACETIC ACID FROM THE ENDOSPERM TO THE SHOOT OF ZEA MAYS L. BY Patricia Lee Hall A large body of literature suggests that a growth hormone precursor moves from the seed to the seedling tip, is there converted to an active hormone, and is then trans- ported downwards to control the rate of extension growth (26). The structures and the concentrations of all of the indolylic compounds that occur in the seeds of corn (Egg gays L.) are now known. Thus, it should be possible to determine which, if any, of the indolylic compounds of the seed can be transported to the seedling in, seemingly, significant amounts. Of interest would be the tran5port of tryptOphan, free indole-B-acetic acid (IAA), and the esters of IAA, which comprise 95% of the IAA compounds. In this work I have shown that, (l) IAA can move from the seed to the shoot, (2) 90% of the tranSported IAA has been metabo- lized into compounds other than IAA en route, and (3) some of the IAA that has moved into the shoot has been esteri- fied. With certain assumptions concerning IAA-flux and metabolism in the shoot, it can be concluded that the Patricia Lee Hall amount of IAA transported and remaining as IAA in the shoot is inadequate to sustain growth. MOVEMENT OF INDOLE-3-ACETIC ACID FROM THE ENDOSPERM TO THE SHOOT OF ZEA MAYS L. BY Patricia Lee Hall A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1977 ACKNOWLEDGMENTS I would like to thank Dr. R. S. Bandurski for his guidance and support throughout this research project and to express my appreciation to Dr. Axel Ehmann for the valuable assistance he so freely offered. I am also grate- ful to Dr. G. Pollack and Dr. N. B. Good for helpful dis- cussions. ii TABLE OF CONTENTS Page LIST OF TABLES O O O O O O C O O O O O 0 iv LIST OF FIGUMS O O O O O O O O O O O O O V INTRODUCTION 0 O O O O O O O O O O O O O l MVIW OF LITEMTUE O O O O O O O O O O O 3 MATERIALS AND METHODS. . . . . . . . . . . 8 Plant Material . . . . . . . . . . . . 9 Application of Radioactive IAA . . . . . . . 9 Tissue Combustion . .. . . . . . . . . . lO Reisolation of Free 3H- IAA . . . . . . lO Reisolation of Free Plus Alkali-Labile 3H-IAA . . 14 RESULTS 0 O O O O O O O I O O I O O O l 5 Total Radioactivity Moved to Shoot and Root. . . 15 3H-IAA Moved to Shoot . . . . . . . . . . 15 DI SCUSS ION C O O O O O O O O O O O O O l 9 LIST OF REFERENCES. . . . . . . . . . . . 26 iii LI ST OF TABLES Table Page 1. Reisolation of 3H-IAA from corn shoots after application to the endosperm . . . . . . l7 2. IAA transport from endosperm to shoot. . . . 24 iv LIST OF FIGURES Figure Page 1. Radioactivity found in the shoot and root of a corn seedling after 2100 dpm were applied to the endOSperm . . . . . . . . . . 16 INTRODUCTION It has been suggested that a growth hormone or a hormone precursor is moved from the seed to the shoot in corn (26). The identity of the transported compound is not known, but could possibly be an IAA-ester, tryptophan, or free IAA. The content of IAA in sweet corn is between 70 to 90 mg of IAA per kilogram of dry kernels (24). In 1941, Avery, Berger, and Shalucha (1) found that more IAA could be extracted from corn kernels after hydrolysis with alkali. Three years later, Berger and Avery (3) showed that kernels of corn contained an auxin precursor that produced IAA on alkaline hydrolysis. Subsequent work has shown that these alkali-labile IAA compounds consist of IAA-myo-inositols, IAArmyo-inositol arabinosides, IAA-myo-inositol galactosides, IAA-glucose, an IAAecellulosic glucan with the glucan chain of variable length, and small amounts of free IAA (15, 23, 8, 18). Ueda (24) found about a 1% per hour decrease in «concentration of all the IAA-esters in corn kernels during germination. This work begins an examination of which compounds, :free IAA or IAA derivatives, can move from the seed to the shoot where growth is occurring. By knowing the rate of movement it may be possible to decide which compounds move at a sufficient rate to account for the IAA and IAA—esters in the shoot. REVIEW OF LITERATURE Cholodney (5) found that a growth hormone diffused out of dehusked 53923 seeds when the seeds were put in hot water or ethanol. When coleoptile tips were placed on one side of a decapitated cole0ptile, a bend occurred which lasted for less than one hour. A piece of the endosperm placed on a cole0ptile produced a curvature which lasted for 3 to 4 hours. Cholodny suggested there was a continual formation of growth hormone in the endosperm and that the growth hormone moved from the seed to the shoot. Skoog (21) decapitated oat seedlings, removed the primary leaves, and placed agar blocks on the coleoptile stumps. He then tested the blocks using the deseeded Aygna test and found no auxin in the agar blocks after 5 to 6 hr, but he did detect auxin when the block had been left on the stump 10 to 20 hr. Skoog also found an auxin precursor that diffuses out of the tip of oat coleOptile sections but not out of the base. He suggested this could be an IAA precursor which is moved from the seed to the shoot. Skoog found the seed to be necessary for regeneration of the physiological tip in decapitated cole0ptiles. Thimann (22) applied IAA in lanolin paste to the scutellum of Axgng_seedlings and observed a slight decrease in growth of the coleoptile due to poorer root develOpment. When seedlings were placed with the roots in an IAA solution neither an inhibition nor promotion of growth of the coleOptiles was observed. Whitehouse and Zalik (27) found that a radioactive compound with the Rf of IAA could be extracted from corn 14C-IAA had been injected into the endOSperm. shoots after They also found minor radioactive peaks at lower Rf's. No vascular strands were found in the endosperm but vascular tissue was present in the scutellum. When roots of seed- lings were placed in a vital stain no dye entered the endo- sperm, but a vascular strand in the scutellum branched and entered the interface between the scutellum and endosperm. Two vascular bundles in the coleoptile were stained. Dye injected into the endosperm did not reach the xylem in the scutellum but did enter between the endosperm and scutellum. Using Phaseolus coccineus, Whitehouse and Zalik destroyed living tissue in a region of the stem with a hot glass rod. l4C-IAA up the stem after injection into The movement of cotyledons was drastically reduced by searing the cotyledons with the glass rod, but die still moved up the stems. This indicates that translocation does not occur in the xylem. Hall 23 31. (12) identified IAA in the phloem by mass spectrometry and in root pressure saps of Ricinus communis. The authors suggest IAA may move more freely in the non- polar phloem. Sheldrake (20) found IAA and alkali-labile IAA compounds in the xylem sap of Zea when the coleOptile tip had been cut off. 1‘ C-IAA injected into 53222 endosperm can be detected in guttation fluid. Sheldrake also sites the autonomous curvature of A3223 coleoptiles as circum- stantial evidence for acropetal movement of auxin in the xylem. This curvature correlates with the asymmetry of the xylem strands at the coleOptile tip. IAA in the phloem could not produce this curvature. A literature review of auxin transport has been published by Goldsmith (9). She states that endogenous and applied auxin move predominantly in the basipetal direction and that this movement depends on living cells. Character- istics of the polar movement are that the velocity is independent of donor concentration and length of section, and that active metabolism is necessary. Goldsmith (10) observed the basipetal movement of IAA in 10 mm sections of oat coleOptiles under anaerobic conditions and found the amount of IAA in the tissue to be significantly decreased within one half hour after anaerobic conditions were begun. In contrast, a significant difference in the amount of IAA that had moved acrOpetally into the tissue under anaerobic conditions was not observed for two hours. Goldsmith also found that only 10% of the IAA that had been moved acro- petrally in aerobic conditions could be rinsed out with large volumes of water while all of the IAA that had been taken up under anaerobic conditions could be rinsed out. She found that anaerobic uptake fit diffusion theory with a 4 mmz/sec. Under diffusion constant of approximately 1 x 10- aerobic conditions, diffusion theory does not hold for movement toward the tip because of immobilization of IAA. This immobilization of IAA explains why basal uptake occurs against a concentration gradient and why the first mm of the section has a higher concentration of IAA then the donor. Goldsmith (11) also found that basipetal transport recycled IAA that had been moved toward the tip. McCready (17) did a series of experiments with 5 mm sections of young, rapidly elongating petioles of the primary leaves of Phaseolus vulgaris L. to determine if base directed and tip directed movement occurred by the same mechanism. He found that low temperature effected the flux of IAA towards the base much more than the flux towards the tip. Inhibitors also had different effects on movement towards the base and towards the tip. Napthylphtalamic acid (NPA) decreased base directed transport until it was equal to tip directed movement. Movement toward the tip was not effected by NPA. Fluorenolcarboxylic acid; 2,3,5-triiodo- benzoic acid; and sodium azide decreased flux toward the base until it was equal to flux toward the tip. At high concentrations of inhibitor, tip directed movement was slightly increased. This could be due to toxic action of the inhibitors which might break down permeability barriers. McCready found no evidence for the existence of an active component in the movement of IAA towards the tip and that the major part of movement towards the tip has the charac- teristics of passive diffusion. It should be noted that in none of the above described work was an isolation procedure for IAA used that has been shown to yield pure IAA thus proving that the radioactivity was in IAA. The method used for IAA isolation in this work, as described below, has been shown to yield pure IAA from Zea (2). MATERIALS AND METHODS Combustion of tissue for determining total radio- activity was done on a Packard Model 306 Tri-Carb Sample Oxidizer. Gas-liquid chromatography was done on a Varian 2740 gas chromatograph with a flame ionization detector and nitrogen as the carrier gas. Ultraviolet spectra were recorded with a Cary 15 spectrophotometer. Bray's solution (4) was used to determine radioactivity with a Packard Tri- 14 Carb model 3003 liquid scintillation counter for C and a Beckman CPM-100 Liquid Scintillation System for 3H. Materials used were from the following sources: 14 2- C-IAA (specific activity 23.5 mCi/mmol) : Schwarz/Mann; 5_3 H-IAA (specific activity 23.5 Ci/mmol) : CEA France, obtained through Dr. M. H. Goldsmith at Yale University; IAA and indole-3-butyric acid : Calbiochem; DEAF-cellulose : Sigma; Sephadex LH-20 : Pharmacia; Silica Gel G plates, Merck Darmstadt : Brinkman; 5% SP-2401 on Supelcoport : Supelco; bis (trimethylsilyl) trifluoroacetamide : Regis; Stowells Evergreen Hybrid Corn 1974 harvest : Ferry Morse Seed Co. Plant Material Corn kernels were surface sterilized, soaked in aerated water for 16 hr, then placed in a horizontal row across a paper towel. The towel was then rolled, secured with tape and placed edge up in a 5 liter beaker containing 400 ml of water. When the beaker was full of towels it was covered with plastic wrap and placed in a dark room at 25 C. Four-day-old seedlings were used for the transport studies. The shoots were between 1.5 and 3.0 cm long. Application of Radioactive IAA About one-half of the seed was cut off from each seedling under a green safelight leaving the embryo, scutellum and about 2 mm of the endosperm intact. Five pl of radioactive IAA in 50% ethanol was applied to the cut endosperm surface, corresponding to 7.2 ng and 2100 dpm when 14C-IAA was used and 7.6 ng and 2,210,500 dpm when 3H-IAA was used. A group of five seedlings, with their cut surfaces facing upward, were placed in a 9 cm petri dish kept humid with moist filter paper. The seedlings were incubated at 25 C for the indicated times, then the shoots, and in some cases the roots, were cut from the kernel and frozen at -20 C until used for oxidation or extraction. Since the endosperm liquifies during germination, this method of application does not require that the radio- active compound permeate any membrane barriers other than 'those which any seedling-endosperm compound would have to permeate . 10 Tissue Combustion Shoots and roots, in groups of ten, were combusted in the oxidizer, the resultant CO2 trapped in an organic base and the radioactivity counted in a liquid scintillation counter. 3 Reisolation of Free H-IAA The isolation procedure adopted is cumbersome and requires almost one week for a single assay. Its advantage is that it has previously been shown to yield pure IAA from seedlings of Egg. Since many IAA adducts and oxidation products have TLC and paper chromatographic mobilities similar to IAA, a rigorous purification must be employed. Sixty shoots were ground with a mortar and pestle in enough acetone to make the solution 70% acetone-water. The homogenate was extracted two more times with 70% acetone and the extracts combined and filtered. Five hundred ul of IAA and 500 pl of indole-3-butyric acid were added. The filtrate was concentrated to 5 ml on a flash evaporator in a water bath at 50 C. The pH was adjusted to 2.5 and the sample extracted three times with 10 ml of diethyl ether each time. The ether was then extracted three times with 5 m1 of l M NaHCO3. After readjusting the pH to 2.5 the NaHCO3 was extracted three times with diethyl ether. The ether was dried and the sample taken up in 1 m1 of CHCl3. For the last experiment the concentrated filtrate at pH 2.5 was extracted into CHCl3 three times instead of the ether, NaHCO3, and ether extractions. ll DEAF-cellulose column chromatography, Sephadex LH—20 chromatography, thin layer chromatography, silylation and gas-liquid chromatography, and UV spectrometry were carried out as described by Bandurski and Schultz (2). The DEAE- cellulose column was prepared as described by Rouser gt 21. (19) for lipids, except that initially the column had to be washed with 100 ml of CH3OH/CH3COOH/(C2H5)3N (20:4:1), then regenerated before IAA would bind to it. The sample, in one ml of CHC13, was applied to the column and the column eluted with; (a) 200 m1 of CHCl (b) 200 ml of CHC13/ 3. CH3OH (9:1); (0) 400 ml of CHCl3/CH3OH/CH3COOH (7:3:0.01%); (d) 500 m1 of CHClB/CH3OH/CH3COOH (7:3:l%). Percentages are v/v. The fractions containing IAA were determined by UV absorption at 282 nm. The IAA was eluted between 350 and 450 m1 of solvent d. The fractions containing IAA were pooled and evaporated to dryness. The column was regenerated with; (a) 200 ml of CH COOH; (b) 400 m1 of CHBOH; (c) 200 m1 3 of CH3OH/CHC1 (1:1); (d) 300 m1 CHC13. 3 The sample from the DEAR-cellulose column was dissolved in 1.0 ml of 50% (v/v) ethanol-water and applied to a 22 x 1.8 cm column of Sephadex LH-20 that had been 'washed with 50% ethanol. The sample was eluted with 50% ethanol at a flow rate of 3.5 to 4.0 ml/hr. Using UV absorption, IAA was found between 80 and 100 ml. The- fractions containing IAA were pooled and evaporated to dryness. The column was regenerated with large volumes of 50% ethanol-water. 12 The sample from the LH-20 column was dissolved in 200 ul of 50% ethanol and applied in a 10 cm streak across a 20 cm X 20 cm thin layer silica gel G chromatography plate. On each side of the sample streak, 1 cm streaks of the sample and guide Spots of IAA were applied. The plate was run in a solvent consisting of benzene-acetone-pyridine 60:39:l. The ends of the plate, where the 1 cm streaks and the guide spots had been applied, were cut off, and sprayed with a color reagent to determine the migration of IAA in the sample. The region.where IAA was present was scraped from the plate and the silica gel was washed three times with 5 ml of 50% ethanol-water. Each time the silica gel was sedimented by centrifugation for 10 min at about 500 x g. The ethanol extracts were combined, filtered through Whatman No. 42 filter paper, and taken to dryness. To remove the small residue of silica gel still present the sample was dissolved in 200 ul of 50% ethanol, transfered to a clean drying flask and redried. The sample was then redissolved in 200 ul of 50% ethanol, transfered to a serum vial, dried under nitrogen at 70 C, and sealed with a rubber t0p. Silylation was accomplished by adding 20 pl of bis(trimethylsilyl)- trifluoroacetamide and 10 p1 of redistilled pyridine to the sealed vial with a syringe. The sample was kept at 45 C for 15 minutes. Gas-liquid chromatography was on 5% SP-2401 on 100/120 mesh Supelc0port in a 1.8 m x 6 mm glass column at 165 C, with 40 ml/min of nitrogen as carrier gas. l3 IAA was first injected into the column to determine its retention time which was typically 15 minutes. A few pl of the sample was then injected to determine the sample pro- file. The rest of the sample was then injected 7 p1 at a time and the IAA was collected by extinguishing the hydrogen flame and slipping a glass tube over the detector outlet. The IAA which condensed on the glass tube was then washed into a quartz cuvette with 1 ml of redistilled methanol. The UV spectrum of the sample was recorded. The 282 nm and the 225 nm peaks were used to determine the amount of IAA present. Absorption by p-coumaric acid was corrected for by multiplying the sample absorbance at 330 nm by 4.0 or 3.3 and subtracting these values from the absorbance at 282 and 225 nm, respectively. The molar extinction coefficient for IAA is 6060 at 282 nm and 33,200 at 225 nm. One half ml of the sample was added to 5 m1 of Bray's solution and counted for 100 minutes in a liquid scintillation counter. A correction for the amount of radioactive IAA lost in the purification procedure was made by using a reverse 3 of the isotOpe dilution assay. The recovery of H-IAA was assumed to equal the recovery of the 500 pg of cold carrier IAA added at the beginning of the purification. The total radioactivity in the corn shoots was calculated from the equation: l4 carrier IAA added (radioactivity of = radioactivity carrier IAA recovered recovered sample) in shoots Reisolation of Free Plus Alkali- Labile JH-IAA The filtered acetone extract from 120 shoots to which had been added 1 mg of IAA and 1 mg of indole-3- butyric acid was divided into two equal aliquots and taken to dryness. One aliquot was taken up in 5 m1 of 1 N NaOH and allowed to stand for one hour in a 100 C oven, then neutralized with H2804. The other aliquot was taken up in l M.NaZSO4. 3H-IAA was reisolated from both aliquots, after adjustment of the pH to 2.5, as described for free IAA above. An additional determination of the amount of IAA recovered was made with a colorimetric assay [(7) and personal communication from Dr. Ehmann]. RESULTS Total Radioactivity Moved. to SHoot and Root The C02 derived from complete combustion of the shoot or root contained radioactivity one hour after the labeled IAA had been applied to the endosperm. Figure 1 shows the amount of radioactivity found in the shoot and root as a function of incubation time. Each point on the graph is the average radioactivity per shoot or root obtained from counting two groups of 10 shoots or two groups of ten roots. After 8 hr, about 30 cpm were found in each shoot. At a counting efficiency of 77%, this represents 1.9% of the applied radioactivity. If all the radioactivity were still in IAA, this would corre8pond to 10 1.4 x 10' g IAA/shoot. 3H-IAA Moved to Shoot The results of experiments in which 3H-IAA was reisolated from the shoot are shown in Table 1. The cpm data were corrected for recovery of the 500 pg IAA added to the acetone extract and for a 33% counting efficiency for 3H. The average amount of IAA that moved from the 15 16 .Eummmooom may 0» omwaoom mums Eon ooam Hmumm mafiaommm cuoo o no uoou com uoocm on» ca oooom muw>wuomoflomm .H whomflm mason _ m w m N H (D l‘ \D “\H , \ “OOH N uoocm x 0H ma 0N mm on mdo 17 kumm mcu oucfl owumuoouooca <flo uo: mm3 uowuuxw 0:0uooo may .N .cofiuomuw mmmnu om .mfimxaouoxc mafiamxam ocm H mucmEHuwmxw :H« mm mma.om mop a.o a.a omnaaoudsn m mmm.aa we m.~ e.m omnaaonoxe non m AH «ma.o~ mea o.a a.v donaaondsn m.m oom.ma «on H.m o.e donaaoudsn soc e e.a ooo.ea we m.~ a.~ donnaondsn m.m mem.oa mN m.H m.a emnaaoudun uoe m AH omH.mH mm h.a omnaaouoan uoc «N as mmm.e~ mom a.a omnaaouomn no: as NHHUEAHOHOU xmww NmN MHOH x uoocm Ammuomuuouv mHmEMm mo wamm uomuuxm mooumod ucmfiflummxm Hum <Hm>oomm unmouom no Homeuomue .Eumomoocm on» on cofluoofiaoom Hmuwm muoocm cuoo Eoum <