Plaint Physiol. (1967) 42, 520-524

Isolation and Identification of .3- (Tryptophol) from Cucumber Seedlings1'2 David L. Rayle3 and William K. Purves Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received January 30, 1967.

Summinary. Crulde ether extracts of green shoots of Culciumiis sativtus L. promoted the elongation of cucumber hypocotyl segments. Puirification of the extract was accomplished by DEAE cellulose, silicic acid, and magnesium silicate chromatography followed by gel filtration and preparative thin layer chromatography. Idelntification of the growth promoter as indole-3-ethanol was achieved bynmass spectrometry, thin layer and gas chromatography, ain( ultraviolet andl visil)le spectroscopy, as wvell as ly physiological characteristics.

The cenitral role of indolic compounIds as plant ctiltnlre meldia containing trvptophan or tryptamine grnowth reguilators is well established. However, (2). Perlex and( Stowe observed lEt production few growth-promoting indolic compotin(ds have been from by 1acilli s cerciis and( by Taphrina isolated in puire form from plaint tissute. The most deforimans (15, 16). \Wightman has reported some extenisively sttidied is indole-3-acetic acid (TAA), evideince for the occuirrence of lEt iII toIllato seed- which has been isolate(d in crystalline form from lings after the applicatioii of tryptophaii or indole- sev-eral sources (5, 10, 20). Toines et al. have iso- 3-lactic acid throtigh the cut stenms (23). In this lated pure indoleacetonitrile from immattire cabbage system it wvas postulatedl that iindolelactic acid was heads (8). Indoleacetaldehy-lde has been i(lentified decarboxvlated to lF,t which was theni oxidized to in plant tissue by Larsen (11). E-idence for the JAA. Libbert aind Brunin have presente(d chroma- occurrence of other growth-promotinig in(Iolic com- tographic (lata showing that lEt -was formed when poutndls, stich as indolepyrulvic acid (18), indole- the TAA-formiing enizyme of peas was inccubated acetamide (1), the ethyl ester of IAA (19), and( for 12 hours in a tryptophani soltutioni (14). others, is based primarily on chromatographic This paper offers evi(lence for the natural behavior of cruide extracts in 1 or 2 solvents. occurrence of lEt in cuctumber shoots. Our method While chromatographic data are valuable, they arc for the purificationi of lEt should l)e applicable to niot concltisive, especially when uise(d as the sole other ineutral inidolic compotuinds. A method is criteria with imptire preparationis. dlescribedl for the bioassay of TEt tusilng the cuctumber The nattural occulrrenice of iiidole-3-ethanol hypocotyl. (IEt) in higher plants has not previously been lemonstrated. Ev-idence lbase(l on paper chroma- Materials and Methods tographic methods and color tests in(licated that 4cetobacterx- ylinm. can pro(ltice JEt. It was pro- RoaSsa(lV. See(Is of Ctcmniis satiZIus L. cv. posed that JEt and( TIAA might arise from indole- Natioinal Pickling (WV. Atlee Btirpee Seed Co.) ace-taldehyde in this organism by a dismtitatiol were soaked for 2 hotirs an(l sown in vermiculite reaction (12). Kaper andl V-el(dstra, tising chroma- (Terralite, Zonolite Co.) moistenied with tap water. tographic techiniques, have (lemioiistrate(l JEt as a Seedlinlgs w-ere grow-n tundler constaint illtumination prodtuct of physiological origini from AgrobacteriumIi (640 ft-c) for 5 to 6 days in a growth chamber at tumefactens (9). Bailey and Gordon reported the 26 to 217°. Segmeints wvere cuit so that the apical tentative identification of lEt in Diplodia llCiS 2.5 cm of the hypocotyl w-as iincludedl as well as the still qutiiescent epicotyl. The cotyle(lonis were remove(l in all cases. The segments were distrib.- tited in lots of ; inito Sten(ler dishes containing ' This work Avas supported by National Scieince Founil- 5 ml of the test medliumni. D)ishes -were returine(d dation grants GB-3248 and GB-4922. conistarnt 2 This material was included in a doctoral tlhesis sul)- to the grow-th chamber and( kept tindler itted by D. L. Rayle to the Graduate Division of the iullumination throulghouit the incubation periodl. Utniversity of California, Santa Barbara. After 4 to 6 houirs the segimienits were measured to 3 National Defense Education Act predoctoral fellow. the nearest 0.5 mm. Hypocotyl segmtents of Cucur- Presenit address: MSU/AEC Planit Research Laboratory, Zuicchili were Michigani State University, East Lansing, Michigan bit(a pcpo L. cv. For(lhook Improved 48823. uised in a similar mianner. 520 RAYLE AND PURVES-IXNOLE-3-ETHANOL IN CUCUAMBER SEEDLINGS 521 Extractiont and Puirification. Seeds of C. saitivus were sown as described above and grown in the light (640 ft-c). After 7 to 8 days the shoots were harvested, weighed, and extracted with ethyl ether -10 (approximately 2 liters per kg fr wt tisstue) for 2 to 3 hoturs at room temperature. After de- 8 cantation of the first extract, the tisstue was ground 0 in a meat grinder and extracted twice more with I fresh ethyl ether. The combined ether fractions 0 were separated from the cellular water and evap- E Z E 0 orated to dryness. The gummy green residue was z z 0 0 resuspended in double distilled water (250 ml per I.- 4 kg fr wt tissue) and filtered throtugh a 4 cm pad 0 z I0 of DEAE-cellulose. This operation ridded the ex- 0 tract of pigments. DEAE-cellulose has the further w advantage of binding IAA. The growth-promoting filtrate was extracted 5 times with equal volumes of ethyl ether. The combined ether fractions were 1 5 9 13 17 21 25 29 33 37 41 evaporated aind the water fractionl discarded. Sev- TUBE NUMBER eral 5 to 15 kg batches of tissue were extracted, FIG. 1. Elution of cucumber factor from Sephadex processed as described above, and stored at 40 until LH-20 by . 10-ml Fractions collected. Selected a total of 93 kg of tissue had been extracted. At fractions assayed for biological activity (4 hr incubation) this time, the residutes from each batch were com- and for absorption at 280 nm. bined to yield 3.21 g of a yellow oil. The combined residues were dissolved in a minimal amotunt of was clamped between 2 Eastman Chromagram petroleum ether (b.p. 30 to 600): ethyl ether (3:1, chamber plates and developed with 100 % CHCl3. v/v) and placed oni a silicic acid column (2.9 X 25 The chromatogram was divided into several zones cm). The coltumn was eluted in a stepwise fashion and the silica gel scraped off and eluted with using increasing concentrations of ethyl ether in anhydrous ethyl ether. An aliquot of the RF 0.15 petroleum ether according to Nlethod A of Hirsch to 0.3 eluate was active in the bioassay. The active and Ahrens (7). The growth promoting fractions eluate was evaporated under nitrogen yielding 2.7 from this step as well as all subsequent steps were mg of material. Final purification was achieved determined by removing aliquiots of the various by rechromatography on Sephadex LH-20 as de- fractions, evaporating the solvent, anid dissolving scribed above. The active fractions were combined the residue in 5 ml of water which was then bio- and evaporated under nitrogen leaving 2.5 mg of assayed. Activity was observed in the 100 % ethyl ether fractions. The active fractionis from the I silicic acid coltumni were combined aind evaporated 100 130 (b) to dryness. The residue (117 mg) was suispende(d A a : in mini-mal amount of CCI CHC13 (3:1, v/v) M+ and placed on a 2.0 X 20 cm magnesitum silicate 30 _ 161 (a) M-1 (Bio-Rad) column packed in CCl4. The Z column was eluted in a stepwise pattern tusing in- .20 _- 4 a creasing conceintrations (0, 10, 20, 30, 40 and 50 %, z 143 v/v) of CHCI, in CC14. At each step, 100 ml of 0 10 4 116 (C) solvent was tused. Finally, the coluimn was eluted 0 l l III I !1 with 250 ml of 100 % CH,CI3. The 100 % OHCI3 II fractions contained the growth-active substance. 4 DO - 130 Further purification was obtained by evaporating -JJl (b) the active fractions to dryness and dissolving the B residue (33 mg) in a minimal amoulnt of absolute z I0- 161 methanol. The methanol solution was placed on a (a) Sephadex LH-20 coluimn (2.5 X /7 cm) and eluted 2 with methanol. Ten-ml fractioins were collected using an automatic fraction collector. Selected fractions were assayed for biological activity and I[I I, ~~~~~~~~~~~~~~116(c) for absorption at 280 nm (fig 1). The active fractions were evaporated in a rotary flash evap- 40 60 80 100 120 140 160 orator leaving 3.5 mg of residue which was dis- m/e solved in and an ethyl ether streaked oni Eastman FIG. 2. Mass spectra. A) Cucumber factor. B) Chromagram sheet (Type K 301 R2). The sheet Indole-3-ethanol. 522 PLANT PHYSIOLOGY a light yellow gum. The progress of the purifica- This fragmentation pattern is characteri-tic of tion was followed by removing an aliqtuot of the simple and can be readily visualized if one active material after each step in the purification assumes that the first electron to be remove(l orig- procedure and spot-chromatographing the residue inates from the indole nitrogen. This characteristic on silica gel in several solvents. Organic com- was of value in the structulral identification of pounds were detected by exposing the dry chroma- 5-methoxyindole-3-acetic acid isolatedl from boviine togram to iodine vapor. pineal glands (13). A secondary fragmelntatioin The purity of the final preparation was checked pathway of cuicumber factor appears to be the loss by thin layer chromatography in 5 solvents of of water from the molecular ion resuilting in thle widely differing polarity: ethyl ether hexane peak at m/e 143. Metastable peaks were seeil at (1:1, v/v), isopropanol NH40H : H.0 (8.5:5:15, m/e 81.5 and 126. The mass spectrum of the factor v/v), CHCI3 : 96 % acetic acid (95 :5, v/v), 100 % was compared with one obtainedl usinig a knowni CHCI3, and CHC13 : methanol : glacial acetic acid sample of indole-3-ethanol (K & K Laboratories). (75 :20:5, r/v). Detection of spots with iodine Prior to analysis, the commercial lEt was purified vapor and with Ehrlich's reagent (2 %, w/v p-di- by thin layer chromatography onl silica gel ilsinig methylaminobenzaldehyde in 2 N HCl in 80 % 100 % CHCI3 as the mobile phase and then re- ethanol) showed the final preparation to be homog- crystallized twice from benzene : petroleum ether. eneotus in each of these solvents. The spectrum obtained for IEt (fig 2B) was similar in every respect to that obtained with the illkown Results growth promoter from cucumbers. IEt and the cucumber factor were compared by A sample of the growth promoting cucumber thin layer chromatography (Eastman Chromagram factor was subjected to mass spectrometric analysis sheets) using 10 different solvent systems. Cor- using an A. E. I. MS-9 mass spectrometer (fig 2A). pounds were detected by spraying with Ehrlich's A strong parent ion peak is seen at m/e 161. reagent and subsequent development at 80'0 for 10 This value is consistent with the empirical formula mintutes. The RF valutes were calctulated and( the C10H1,NO. The main fragmentation pattern of the Ehrlich reaction colors noted (table I). Cuiculmber parent ioln is rationali7ed as follows: factor and IEt were also compared by gas chro- matography, using an Aerograph Model A-90-P3 1201 H, [ HCH2 (column: 20 % SE-30 on Chromosorl) N76W)/80, 5 ft X 0.25 in, coluimn temp 2100, detector temp -OCH3 2800, injector temp 2800, carrier gas heliuim at 60 ml/min). Under the conditions employed, reten- tioIn times of 6.94 mintutes were recorded for both a) m/e 161 b) me a 130 IEt and the cuicumber factor. In contrast, the ethyl ester of IAA had a retention time of 10.51 minutes. Dedio and Zalik have recently separated certain -C2 volatile indoles inclu1ding IEt uising similar conidi- tions (3). Figure 3 shows the ultraviolet absorption spec- tra of IEt and the cuicuimber factor. Absorption A maxi-ma appeared at 274, 281, an(d 291 urm in both cases. The spectruim of IAA is incluidedI for com- C) rne a 116 parison. Harbo aln(d Aasheim iused th2 visible 'Fable I. RF Values for Cucumber Factor and Indoleethanol in Silica Gel Thin Laver Chrmwatography Eastman Chromagram sheets were used for these determinations. Cucumber Solvent factor Indoleethanol RF RF Hexane: ethvl ether (1:1, v/v) 0.18 0.20 Hexaue: ethyl ether (1:4, v/v) 0.59 0.62 Isopropanol: NH,OH : H,O (10:1:1, v/v) 0.99 099 CHC13 : methanol: glacial acetic acid (75:20:5, v/v) 0.98 0.99 CHCI methanol CC14 (50:25:25, v/v) 0.81 0.80 CHC13 methanol CC14 (5:4:4, v/v) 0.99 0.99 CHCI3 0.20 0.20 CH2Cl2 069 0.70 1- ethalnol: H,O (40:4:19, v/v) 0.92 0.92 Ethanol : CCI4 (1:1, v/V) 0.99 0.99 RAYLE AND PIURVES-INDOLE-3-ETJHIANOL IN CUCUMBER SEEDII NGS 523 ever, the test samples were so small as to give spectra uinsatisfactory for publication. Dose-res,ponse cuirves for the factor and lEt in the cuicumber hypocotyl test are given in figure 4. The response to optimal IAA is shown, for com- parison. In outr preliminary studies of the factor it was noted that, while some species responded strongly, others responded weakly or not at all. Zuicchini squash was one of the species that did not respond significantly to the factor. lEt was also inactive in the sqtlash hypocotyl test (fig 5).

8 E E 0 WAVELENGTH, nm FIG. 3. Ultraviolet absorption spectra of cucumber CD4 - factor, indole-3-etlhanol, and indole-3-acetic acid in an- z hvdrous ethN7I ether. Concentrations of cucumber factor and indoleacetic acid adjusted to give approximately equal w absorbance. Concenitration of indoleethanol adjuisted for 2 loxN-er absorbance to facilitate comparison. spectrum of the colored produtcts of the action of Ehrlich's reagent on IEt as evidence for its occuir- 0 10-85 1i5 10o4 103 rence in culture media of Acetob(acter xvlinumn (6). \Ve compared the visible spectra of cucumber lEt, M factor and of IEt after the addition of Ehrlich's FIG. 5. Dose-response curve for indole-3-ethanol in reagent. The spectra were identical, showing sharp the s(luaslh hypocotyl test. Measurements made after 6 peaks at 575 and 545 nm with a small peak at 460 hours. Bar indicates segment growth in 0.1 mm IAA. nm and a large, broad peak from 395 to 420 nm with a maximum at abotut 4405 nm. Infrared and ntuclear magnetic resonance spectra of small sam- Discussion ples of the cucumber factor were also obtained. These corresponded to those of known IEt; how- This paper presents convincing evidence for the occurrence of IEt in cucumber seedlings. The mass spectra of lEt and the niatturally occturring 14 produLct are identical. The factor and MEt co- A chromatogram on silica gel in 10 solvents of widely 12 differing polarity and have identical retention times E 10 in gas chromatography. The tultraviolet absorption spectra of the factor and IEt are identical as are z 8 the visible spectra of the colored products of re- O actions with Ehrlich's reagent. The ctucumber fac- 06 tor and IEt elicit similar growth responses in the 4 cuctumber hypocotyl test and 1)oth are inactive in the squash hypocotyl test. 2 The structure indole-3-ethanol is favored over

0 indole-2-ethanol. Various 3-substituted indoles such 0.025 2 3 4 0.025 2 3 4 as IAA are known to occtur in plants, while no I E t M/m C F . g/4n likely prectursor of indole-2-ethanol is known. The physiological response caused by the factor is iden- FIG. 4. Dose-response curves in the cucumber hypo- tical with that cauised by known inclole-3-ethanol. cotyl test. A) Indole-3-ethanol. B) Cucumber factor. Measurements made after 20 hours incubation. Bars The reaction with Ehrlich's reagent also suLggests indicate segment grow-th after 20 hours in optimal 0.1 the structure indole-3-ethanol, since the Ehrlich tnM IAA. Experimnenits A and B performed oni dif- test is characteristic of indoles in wh ch the 2-posi- ferent days. tion is unsubstituted (4). 524 PLANT PHYSIOLOGY IEt has been reported to be slightly active in of silicic acid1 chromatography T. Bi(,.C. lihem. the Avena and pea tests (21) and moderately active 233: 311-20. in the wheat cylinder test (22). However, the 8. JONES, E. R. H.. H. B. HENE,IS.r (J. F. SMITHi, analogs of the knowni acidic auxins have, %NDn J. A. BENTrIEN-. 1952. 3-Indolylacetoniitrile: a naturally occurring l)kant growtxh hormaonle. Na- in general, been very little investigated. Interest- ture 169: 483-87. ingly, JEt is as active as IAA in the cucumber 9. KAPER, J. M. \.\1 H. VE-LDSTRA. 1958. Onl the hypocotyl assay. We have found JEt to be an metaboli.Ksm of tryp)tol)llane 1h 1 (J)w'kactcriuml tit- active growth promoter in certain species other umcfaci,ins. Biochilm. Biophys. Acta 30: .401-22. than the cucuimber. These data will be reported 10. K(.L, F1. ANI) I). G. F. R. KOSTERMIANS 1934. separately. Hetero auixin al s StoffNx-ech.selproduk-t n iederer One may ask whether lEt is itself an active ptflanzlicher Orgaiiisiiien. Isolierung *lns Hefe. growth hormone or whether it is coniverted to IAA Hoppe-SeylerI Z. Plhysiol. Clleim. 228: 113-21. 11. LARSEN, P. 1951. Formationi, occulrrence and in- before it is active. \Ve are not yet prepared to activation of grow th suibstanlces. _-iln. Rev. Planit answer this question; however, a 2-step oxidation Phvsiol. 2: 169-98. of IEt to IAA via indoleacetaldehyde seems likely. 12. LARSEN, P.. A. HARBO, S. KLUNOGSiYR, AND. T. The interaction of IEt with IAA in the cucumber AASHEIM. 1962. On the biogenesis of somiie indole assay is consistenit with this view (17). This quies- coml)ounds inl 4 cttobactc'r xrvlimml. Phvsiol. tion is currently iineder investigation. Planitarumll 15: 552-65. 13. LFRNER, A. B., J. D. CASE, K. BIEMANN. R. V. HEINZELMIAN, J3. SZ1\iuSZKO\-IeZ, XV. C. ANTHONY, Acknowledgments AND A. KRIVIS. 1959. Isolationi of 5-methoxvin- lole-3-a,etic acid froiim bovine pineal glantls. J. We thank Prof. David Lightnier of UCIA for making Am. Clhem. Soc. 81: 5264. personnel and facilities available for the mass spectro- 14. LIBBERT. F. AND K. BRUNN. 1961. Nachweis x'On1 metric analyses. We thank Prof. Bernard Baker for his Indol-3-breniztraubenistiure undl Indol-3-athanol valuable suggestions. Larry Vickery anid George Strong (Tryptophol) bei der enzvimatischen A.uxinbildunIg provided skillful technical assistance. aus Trvptoplhan in vitro. Naturwxissenlschaften 48: 741. Literature Cited 15. PERLEY, J. F. AND B. B. STOWE. 1966. Oi1 the ability of TJaphrina deformians to produce inidole- acetic acid fronll tryptophan by way of tryptamine. 1. ANDREAE, WX. A. AND N. E. GooD. 1957. Studies Plant Physiol. 41: 234-37. on 3-indoleacetic acid metabolism. IV. Conjuga- 16. PERLEY, J. F. AND B. B. STOWN-E. 1966. The pro- tion with aspartic acid and ammonia as processes luctioni of tryptamine from tryptophan by Bacillus in the metabolism of carboxvlic acids. Plant ((crcis ( KV-T). Biochemii. J. 100: 169-74. Physiol. 32: 566-72. 17. PtURVES, XV". K., 1). L. RAYLE, AND K. D. JOHN-SON. 2. BAILEY, G. B. AND A. C. GENTILE. 1962. Indole Actions and(l interactions of grow th factors on cu- compounds synthesized by Diplodia natalensis. culmber hypcwotyl segmenits. Annii. N'ewv York Planit Physiol. 37: 439-45. Aca(l. Sci. Ini p)ress. 3. DEDIO, W. AND S. ZALIK. 1966. Gas chronmatog- 18. STONVE, B. B. AND K. V. THI-MANN. 1953. In1dole- raphy of indole auxins. Anal. Biochem. 16: 36-52. pyrui ic acitl inimaize. Nattire 172: 76-66. 4. ELDERFIELD, R. C. AND B. A. FISCHER. 1958. Als- 19. TEUBNER, F. G. 1953. Idenitificationi of the aulxins tonia alkaloids. IX. Synthesis of alstonilinol and presen1t in apple enidospermn. Scienice 118: 418. analogs by reductive ring closure. J. Org. Chem. 20. THIMANN, K. V. 1935. On1 the plant grow th hor- 23: 945-53. monie pro(lulced by Rhivo)pius suiinus. J. Biol. Chem. 5. HAAGEN-SMIT, A. J., W. B. DANDLIKER, S. H. 109: 279-91. WITTWN'ER, AND A. E. MURNEEK. 1946. Isolation 21. VELDSTRA, H. 1953. The relationi of clheimiical of indoleacetic acid fronm immature corn kernels. strticttire to bliological activity in groxlth stib- Am. J. Botany- 33: 118-19. stances. Anin. Rev. Plant Physiol. 4: 151-98. 6. HARBO, A. AND T. AASHEI-M. 1962. A spectropho- 22. XXIGHTrMAIN, F. 1962. -Metabolism and biosynthe- tometric method for the identification and assay of sis of 3-incdoleacetic acid and related indole com- tryptophol and other derivatives. Phvsiol. Plan- pouinds in planits. Can. J. Botanx 40: 689-718. taruini 15: 546-51. 23. XVIGHTI.AXx, F. 1963. Path-ways ot tryptophan 7. HIRSCH, J. AND E. H. AHRENS, JR. 1958. The metabolism in tomato plants. Collo(l. Intern. Centre separationi of comn)lex lipide mixtures by the use Nat]. Rechl. Sci. Paris 123: 191-212.