ether-soluble & ether-insoluble auxins from immature corn ... · ether-soluble &...

Ether-Soluble & Ether-Insoluble Auxins from Immature Corn Kernels1,2B. I. Sahai Srivastava

Grain Research Laboratory, Board of Grain Commissioners for Canada, Winnipeg 2, Manitoba

IntroductionAuxin activity in plant extracts is attributed to

IAA and otlier ether-soluble auxins. The ether-insoluble auxins are usually ignored, although ether-insoluble auxins that liberate IAA and IAN3 onalkaline hydrolysis have been described (1). As-corbigen, a derivative of ascorbic acid and IAA,which y-ields IAA on alkaline hydrolysis, has beenreporte(d to occur naturally in Brassicae and otherplants (6). Wightman (24) has recently reportedthat a compound resembling ascorbigeil was formedin the metabolism of tryptophaile by the cabbageplant. Steward and Shantz (22) have suggestedthe occurrence of IAA-arabinose in immature cornkernels andl Zenk (25) has characterized IAA-glu-cose as a metabolic product of IAA in Colchicumleaves. Srivastava and Shaw (18) have reportedthe occurrence, in several plants, of ether-insolubleauxins that were active in the Avena growth test,andI wlich liberated IAA on alkaline hydrolysis.Farrar (5) has recently described the occurrence ofseveral ether-insoluble auxins in iiliimature cornkernels. The present paper deals witi the study ofether-soluble and ether-insoluble auxilis iil imilmaturecorn kernels.

Materials & MethodsFreshly harvested, immature (early milk stage)

corn kernels (Zea ;niavs L. var goldlen beauty))wereused for the extraction of free ancI bound auxins.

Free auxins are regarded as auxins (2, 6, 8, 12)actually present in a sample of plant nmaterial at agiven moment that should be extractedl by procedureswhici exclude chemical or enzymic clianges duringextraction. Bound auxins, on the otiler hand, areregarded to exist in plants in many fornis (2, 6, 8,12) such as IAA-protein, tryptopilane-protein, andinterconvertible auxins. All of these may be con-verte(l to IAA by hydrolysis, enzymolysis, or autol-ysis. On extraction of the plant tissues \vith etherat approximately 250 the bound fornls of auxins be-

1 Received Dec. 24, 1962.2 Contribution No. 218 from tile Grain Research

Laboratory, Board of Grain Commissioners for Canada,Winnipeg 2, Manitoba, Canada.

3 Abbreviations used in this paper: IPyA, indole-pyruvic acid, IAN, indoleacetonitrile, IAMI, indole-acetamide, TNH,, tryptamine.

come convertedl to IAA. This conversion is presumi-ably caused by enzymic activity wvhich persists (lur-ing the extraction.

Free auxins were extracted with coldl absoluteethanol whereas the bound auxins w-ere extractedIwith ether followedl by extraction of the residlue withcold 90 % ethanol. The latter procedure was adopt-ed for the extraction of bound auxins after it wasfound that ether-soluble and ether-insoluble auxinswere not completely extracted with ether and that90 % ethanol coul l extract residual amlounts ofthese substances.

Col(d Ethanol Extraction. The kernels wx-ere sep-arated froml the stalk, frozen wvith dry ice, homog-enized with dry ice chilled absolute ethanol (10 11lethanol/g fr wt kernels), and extracted overnightat -10° in the clark. Snmaller samiiples (ca. 10-30g fr wt kernels) were extracted for the quantitativeesimation of auxins whereas the larger samiiples (ca.100-500 g fr wt kernels) were used for the isolationand identification of auxins. Alcohol was selecteclas the extraction solvent since it inactivates nlost ofthe enzymes ancl is an excellent solvent (12) for theextraction of auxins. If plant tissues are extractedwvith water, acetone, ether, or cliloroformii enzymlicactivity persists clurilig extraction; aili the additionof enzyimle iinllibitors (luring extraction has nIot provx-ed useful (12). The extraction with alcollol has ledto the production of artifacts (13) an(l Fukui et al.(7) have reportecl thiat esterase activity persists onextraction of plant tissues with etilaniol. Nevertie-less, in the extraction procedure used in this labora-tory, where a fairly large aniount of alcolhol wasused and the extraction was carried out at low tenm-perature the production of artifacts clue to enzymicor cheillical reactions if not excluded was certainlymininmized. The author has reported (17) that IAAadded during extraction of plant tissues uniler theseconditions was recovered aliliost quantitatively.

The alcolhol extract was concentratecl at 350 invacuo. The resi(lue was taken up in water (10 mlw-ater g fr xvt kernels) at 250 and filtered tllroughcelite to renmove precipitatednmaterials. The filtratew-as adjusted to a pH of 8.5 with 5 %cSNaXCo3 andshaken three tinles with peroxidle free etlher (12) toyield tile ileutral-etlier fraction. The filtrate wasnext adjustedl to a pH of 3.0 w\ith 0.5 N HCI andshaken three tiilles with ether to yield the acid-etherfraction. The aqueous phase left after ether extrac-tion w,vas termzed the aqueous fraction. If the sep-aration of acid and neutral ether soluble substances

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ilnto txvo fractiollns Wasniot desired the filtraite wNasdirectly adjusted to a pll of 3.() \vith O.5 N HCI cInshakeln thlree timies with ethier to vield acid-neutraletlher fractioni. The etlher fractions wvere alxxvavswvaslie(d \-ithi water, (iriei( ovel- anlihv(irous Na-S04.filtered, ani( evaporated to dryness in vacuo. Theacqueous fr-actioln wvas concentrated in vIcuo at 350.The authior has fouid that, if the ethier fractionswvere w ashed wvith xvater an1(l (Irie(I ovel-r anhvdrousNa.I S plriOr to paper chlroImatogra-ph)yIV thle conl-taminants like sugars ol anililo acids \v ere not dle-tecte(i on1 the chromlatogorams.

The various fractions xwere chromatog-graplhed as

spots or ban(ls on \Whatman 'o. 3 i\rI chirolimat-ographic paI)_er. Chromatog-ramsx vere dlevell)pedin glass taniks at 22 + 1° in the dark generally \vithisopropanol: H.,O (10: 1) in an atmosphere e(Iuili-brated wx-ith isopropanol H 0: NH40H ( 10: 1: 1 )uniless imientionie(l otherxx ise. I)escending clhromaI.Lto-grap)hyxwas usedl througilout andi the chromatogramisafter dlevelopIment xvere eithiel- used for l)ioassav orfor furthier purification. isolatioln. Can(l i(lentificatiOnof auxills.

Ethier fractions e(qui-alenit to g of freshi \eightof kerniels and the aqueous fraction equivalent to 1g of freshi x eight of kernielsx ere normally chlro-mlatographie(d for the bioassax of aUxinlls. For- bio-assa- chromatogranis xvere (livi(led into ten e(iualsegmiielnts. Each segmilenit xxWas then elutel \v ith 5ml of (leioniize(l (listille(d \vater and the eluaites aftersuitallie (lilutions (100-fold, of auxins I)resent inlarge amounts ) xvere assayed in the Avena gro-oNvthtest using Brighton Oats as (lescrilbedl hv Shax\ andHax kiins (15) andl Srivastava al Sl x(s19 )Distille(l wxater colntrol and I AA stamdards (0.01,0.1 & 1.0 mig liter ) xvere inclu(le(l xvithi each runii andthe amounts of various auxinls xvere calculated asIAA eqiuivalenlt fromil the standard curve of IAA.Booth (3 ) hals reported that Sugairs p)roiote eloniga-Lioin of coleoptile sectionis ai(l nmay thierefore mimiiicatxin,Visxvhen hioassav i odioe in (listilledl xater. Inthe l)resent fractioncation I)roce(lure the ethel- frac-tionssxvere obtained free frolmi sugars and amilloacids. The sugars \x ere only (letecte(l in the upperone-third part of chroinatogram of the aqueoum frac-tioln. The auxills present in that regioni xvere elute(dfor hioassay \x ith a lar-ge amount 'of xvater (500 mlH ,g fr \t kernels) so that the concentration- ofsugars in the eluate \xas niot high enough to causesignificant timiulatioln. To cause anx significantstimulation of coleoptile Sections 1 to 21% x>vas the

inimumil1ll colncenitrationi of sui-ars required ( 3, 4)ainid stimiulatioln of coleoptile sectionis by 0.1 to 0.2 c%,sucrose xxas x irtuallx the slaime as in (listillecl xxater(3 . Nevertheless, the inter-felrenice fromi stugairsand amino alci(ls (lue t) their- synergism xvith auxillsainI thle initelrferenlce fr-omll unknixvnI inhibitory sub-stainces xvhich miiav occtU in the smiie zone as IuXinSis, ot cour-se. p)ossible. Therefore, in conisi(ler-inigthe quantitative (lata l)resente(l in this pial)per. the

limitaltion"s of the hWioaSSax technlliques shoulld be keptiln mind.

For the idlenitificationi of valriou comilpoulid's. tilechromatograms xx-ere lsprayedl xx ith various clhromlo-genic reagents. The chromatog ramis Sprayed xxwithEhrlichi Salkoxski ('12 ). nitrite-nitric acid. 2.4-clinitrol)lpenvIlhvd(razinie hvdrochlori de ('16 ), anil am-mon(iiiacal silx'er nitrate ( 2 ) xxee (develolpedl (t roomtemperature xxwhereas the chronatogranis sl-pra1vedxxwith niinhvdrin-acetic acid, ninhvdrin, (liipheny llilliephosphate, and aniline pithalatc' ( 16 xx ere de vtl-opecl at 1000.

Attemlpts xvere imad(le to purify the auxinslr)e.senltin the aqueous fraction by adsorptioll onl stearic 'Icidltreate(l charcoal (16 ) folloxxe(d v elutioni wxith 8a(Iueous phenol. Since the recoverv of aixins fromthe chaircoal xasl)ooir al 11d0iisu)bstantill l)ui'ificatioilNxvas achieved. this stel) in the plulrificatioil plrior to

paper chromatography I1 isopropanlol-ammonllia sol-vent xas abaln(lonie(d in favour of rechromatog rli) v

of in(li i(lvidal auxin zones ill 11-blutanlol acetiC aciCdxvater ( 4: 1: 5 ) Rechr-maitog-raphy, x here nieces-sarv, wx'as also uised in the purification of auixillns ofethier fractions. After p)urificatioil the aixins xx ereeluite(d fr-olim the paper by appropriate sol-xents ( etherfor ethiei- fractionl a1lxillns aid xxwatei for auIeteousfractionl iuxillns ) andl(l the ultraviolet sp)ectr'a of thleeluates xxere (leter-inilie(l usinga< Bleckmall ( \[odel1L ) sl)ectroplhiotometer. After the (letermilinationof spectra. eluates containing auIxinis of the 'iqueoosfr-actioln xere hydrolyzed wx ith ilkiali. 'I'le hvdroly-sates xere examined, by alpl)rol)piiate methods, foi- theplrodlucts forimie(d as (lescribe(d beloxv.

Ethcr -Ethizool E.ixtrction. Kerinel s fromi thesame col) xere uxsedl for the extraction of firee andboundl auxins. Ten to 30 of kerilels xere extract-ecl at natural tissue 1)H (7.0) xwith l)eroxidle-freeethier (10 mil etielr g ker-lels ) fol 48 hours at 250in dar-k by placing the contents ini a flas-k on a shak-er. The ethier- xas changed once (dur1-lig the extrac-tioln. The ether- extracts x erc pooled a1ln the rc'si-(Inue xlvs extr-acted xxith c 1'1 90 ", ethanollu ] 'vern-ightat - 10(. The 90 (( ethanol extract and the ethierextriact xx ere conilnined aldl concentrated at 35 in'actio. The resi(dule xwas takeln upl) in xater ( 1( mlH.g,,g fr wxt kerinels ) at 25i and filtered thiroughcelite. The partition of the filtrate inlto acid-neutralether alld alaueous fractions an Ili- the subsequent c¾lro-matography xwas carriei( out as described earilier.Usually tilree to four chromnatograims xere preparedfromii the ether-soluble and etlhe illsInul+' fractions.One of these x .as tested by Avenia bi s11 (I1V, t\\xxvere slIrayecd x ith Elii-lichi aI Salkox ski reag'entsalld from1i tile fourithil chromatograili of aci'1-neuitralether fraction the IAA reg ion )f tile chrIOmIaItt-og r1aIm\xxas elute(d xxith ethier and the tiltraxvicdl-t spectrumof the eluiate xws (leteriniiie(l.

ResultsCol(d Ethaowl Extr-o(t. R,K values, colo reiac-

tionis, maximia,and1inlinin.a of thCe uIltra,Violet Sp)ectIL.,

474

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SRIVASTAVA-AUXINS FROM IMMATURE CORN KERNELS

and the concenitration of the compounds detected inacid-ether, neutral-ether and aqueous fractions of thecold ethanol extracts of immature corn kernels arepresented in table I.

Auxins in Ethler Fractionzs. Chromogenic re-agents usually produced five to six spots on thechromatograms of acid-ether fractions, and three tofour spots on the chromatograms of neutral-etherfractions. Compounds with Rf values of 0.35 andl0.40 from the acid-ether fraction always producedthe most pronminent spots. The compounl with Rfvalue 0.40 was active in Avena bioassay, and wasidentical in color reactions, Rf values in four sol-vents (Rf 0.70 in acetic acid: water, 1: 3: Rf 0.82in n-butanol: acetic acid water, 4: 1: 5; Rf 0.84 inisopropanol: wsater, 10: 1 in the absence of am-monia; an(l Rf 0.40 in isopropanol: water, 10: 1 inthe presence of ammiiiionia), and in ultraviolet spec-trum (Xmi,, 245 mut, Anlax 280 miu, Shoulder 290 nlmi)with authentic JAA (X mi n 245 mI, XA max 280 mit,Shoulder 290 m1U). This eviclence indicates thatthis compound Nas indeed IAA.

The compound with Rf value of 0.35 that produc-ed green color with Ehrlich's reagent decomposedon chromatography in isopropanol-ammonia solventinto two compounds with Rf values of 0.57 and 0.75that gave blue-purple colors with Ehrlich's reagent.Schwarz and Bitancourt (14) have reported thatindolepyruvic acid in enol form gives with Ehrlich'sreagent a yellow color that turns later to green.The ultraviolet spectrum of the compound with Rf0.35 (X min 230 mA., X max 250 miu, Shoulder 285 nitu)however, resemnbles wsith the ultraviolet spectrum of3-methyloxindclf (Xmin 230 nitu, Xmax 247.5 nl/,,Shoulder 275 mn) (9) indicating that it may havean oxindole structure. The compound that gavegreen color with Ehrlich reagent was not detectedwhen acid-ether fraction was chromatographedin acetic acid: water (1: 3) or in isopropanol:water (10: 1, in the absence of ammonia) solvents.Instead, a compound that gave a yellow color withEhrlich's reagent was detected at Rf 0.72 in aceticacid-water solvent and at Rf 0.74 in isopropanol-water solvent. Except for IAA the nature of othercompounds detected in the acid-ether fraction is notknown. The comlpounds with Rf values of 0.52 and0.80 in the acid-ether fraction may, however, hesimilar to X, and X. respectively of Stowe andThimann (23).

In an attemlpt to resolve the controversy regard-ing the presence of IPyA in maize kernels (2, 10,23) the acid-neutral ether fraction was chronmato-graphed in three solvents (acetic acid: water, 1: 3;isopropanol: water, 10: 1 in the absence of amnmonia;and in isopropanol: water, 10: 1 in the presence ofammonia). Since IPyA is relatively stable in acidor neutral solutions as compared to alkaline solu-tions (2, 10, 14) the extracts were not permittedl tobecome alkaliine during extraction or fractionationand the whole process of analysis was completedwithin 2 days. The chromatograms were sprayed

wN-ith Ehrlich, Salkowski, ammoniacal-silver-nitrateand 2,4-dinitrophenylhydrazine hydrochloride re-agents. Immediate reduction of AgNo3, reported asa test for the enol form of IPyA (2, 14) and reac-tion with 2,4-dinitrophenylhydrazine hydrochlorideas a test for keto form of IPyA (14). xvas not ob-tained on the chromatograms developed in the threesolvents used. Brox-n to black spots indlicating thereduction of AgNo3 were, however, produced 3 to 5minutes after spraying the chromatogramls with anm-moniacal-silver nitrate. These spots had Rf valuesdifferent from those reported for IPy A, (2, 10, 14).Browvn to black spots xvere procluced (lue to the pres-ence of phenolic conmpounds particularly ferulic acidin the extract. Since pure IPyA xvas not availablefor a comparison of its behavior under the author'sconditions no definite conclusion regarding its oc-currence in corn kernels can be made.

In the neutral-etlher fraction (table I) no evi-dence for the presence of TAN an(l TNH., xvas ob-tained since the nitrite-nitric acid and ninhilvldriln-acetic acid reagents xvhich react xvith IAN andlTNH, respectively to produce characteristic flu-orescence under ultraviolet light did not give thesereactions. The compouncls of this fractioni reactedwvith 2,4-dinitrophenylhydrazine indicatinig that theymay contain aldehylde or ketone groups. Theirnature, is hovever, not knoxvn.

Au.rins ini the Aqutcouts Fractiont. Six auxinsthat stinmulate(d growth of Avena coleoptile sectionswvere detected with the chronmegenic reagents in theaqueous fraction of the cold ethanol extract of im-mature corn kernels. Their concentration, Rfvalues, chromogenic reactions and the miinima andmaxima of their ultraviolet spectra are presented intable I. They produced characteristic indole colorswith Ehrlich and Salkowx-ski reagents but were nega-tive to ninhydrin and aniline phthalate. Their Rfvalues in n-butanol: acetic acid: water (4: 1: 5) sol-vent xvere fairly close to those obtained wvith iso-propanol-ammonia solvent. All these compoundswere adsorbed by stearic acid treated charcoal (16)and were eluted with 8 % aqueous phenol. Duringthis process some decomposition occurred and re-covery xvas generally lowv. Since the auxins of theaqueous fraction could not be extracted wvith etherfrom their chromatogram eluates at acid or alkalinepH none of the compounds corresponded to IAMIsince it is reported (25) to be formed during chro-matography of labile auxins in ammoniacal solvents.As judged by the intensity of the color reactions andthe results of Avena bioassay the auxins with Rfvalues of 0.26 and 0.39 xvere present in largestamounts in the aqueous fraction.

The ultraviolet spectra of the auxins of theaqueous fraction showed indole like features, namiiely,minima at 240 to 250 mit, mlaxima at 270 to 285 mijand shoulder at 290 mit. The spectra Nere, however,slightly different from those of pure indlole com-pounds. In order to investigate further the natureof these auxins, the aqueous fraction from approxi-

475



Table IR,I ! ailucs, Color RIeactions, Spectral Cha rachcrs, &' the Conccntration of Substances Prcscnt in Acid-Ether,

XciVtral-Ethcr, (-, Aqucous Fractions of Cold Ethaniol Extract of Inoniiiatutre Cornl Kerncls

\Ieaii*FIraction R,*

N-altElirlicli Salkow ski Niilhydrin-acetic acid

Nitrite- Dinitro-nitric acid phlienyl-lly(lrazine

SpectralCharacters

Max m,uAg ill 1ug

MeanI conc-'**inlg equliv

1A\ k(g fr xvt

0.00 Blue0.21 Purple0.35 Green0.40f Purple(0.85)0.52 Purple0.8() Purple

0.63 Oranige0.70 Crimson0.86 Purple

0.15Y) ellox-(0.11)

0.26 Purple(0.30)0.39 Purple

(0.43)0.47 Purple

(0.51)0.6.3 Purple

(0.70)0.75 Purple(0.7o)

Pillnk

C rimison

(rillisollGreyCrimiisonGrexGrex

-groween Blue-purple

Purple-grey

C rillisoll

0.93250 230280 245 . 3.90

Pink PinkPurple-orange PiikYelloxw P'urple

... Purple

. C rilliso

Crllnison

Pink

(rilll.soI

YellowYellowBroxx n

275-285 240 0.13

275 247 42.00

265-270 250 45.00

280 252 (.2)-... ... 270 250 ((.64... . 27 8 248 ().42

AlMean of fix-e (leterminiation1S.R value., in isop)ropanol w:l:ater (10: 1) in ani atmlosl)lpere saturated with isopropanol :lroxi(le ( 1(: : 1) RK Values in brackets in nt-butan ol: acetic acid : water ( 4: 1: 5).Conlcentrationis (letermiiec(l bv Av-enia hioassav from

t Id(lenltifiecl as I AA.

matelyv 500 g kernels xxwas chromlatogral)lle(l as balds

in isop)rol)pinol-amiiilil-)luial solvent alid the varniousatuxin zonies xwere eluite(d xvith xvater. Thle eluiatesfroni Rf valutes of 0.15, 0.47. 0.63. and(I 0.72 Nx ere

rechromatographed iniisopropanol ammonia sOlvxentxx hereas those froml RI' values of 0.26 anld 0.39 xx ererechromatographied inl i-hutaniol: acetic acid : x ater

(4: 1: 5") solxent. The rechromatographed eluates

x were hvdlrolvzel hv autoclavinig at 1000 xwith 0.5 _N

Ba(OH )., for 30 to 60 miniuttes. The hNvdrolvsatesxxere cooled, ad'justed to pH 3.0 xitlh l- H.,SO, andfiltere(d throughl celite. The filtrates xere l)artition-e(d ilnto ethier andl aqtueous fractin,,;is 1y shaking ithethier. Ethier fraction fr-oni each sanmple xxas xashed

ith xxvter, (drie(d oxer alnhvdrous NaL.SO,, and

chromllatographed as tlhr-ee aliquots in isoprop,110ol-aimmonia solv-enit. T'lhe chromataogramiis of the twxoaliquots for each sample xecer spraved xvith Elhrliclialn(d Salkoxski reag,-enits. TIlhe .\A zoile of thetlhirdl aliquot xxas eluite(d ith etlher anid the ultra-violet spectrumii of the eluate xvas taken. The (le-x-elopmiielnt of chromogeniic rieactin)iis similar to andlat the same RI xvalues as IAA 1x)-o\e l the formation

of IAN on hydrolysis of aqueous-fraction aluixills.Thle ultravivolet sl)ectra of 1A\A. ( A,,,i 245 XA,,X

wx ater: anininyoilntiuiu hiy-a IXAA stanidardr curve.

280 my, Shoulder 29) mii,u thu,s rolducedl. w cr

very similar to the sl)ectrum of aiutilenitic IA-A\ ( A0245 my, XA,lX 280 rug. Shlouil(lei- 2)0) iiy ).

The aqItueous fraction firomii the h ydroPs-oates o0the a(queouts fractioln auxinis as concentrated at 350

inI Vacu all c(hrolriato-raiphed in 11-butall! '1: aceticaci(l: xater (4: 1: 5)' eth\ l acetate: )yri(diine: x-ater(10: 4: 10) ain( isoprl)Ianol-liiioia solxuents.Glucose and( arabinose ere runtii as mlal-ker-s and(the chironmatogramilsx -ere sp)raye(l wxith ninhydrin anld(anilinie phthalate or (diphenylaniine phosphate. Noexvidlence for the 1iberati,n oft amino acids xs obh-tainie(l. Spots haxingo the same R, valute and gixing

the same color realctionis as arabillose xere onyplro-dluce(l after hydrolysis of a(lueous fractionl aIuxillof R, xvalue 0.39. The positive chromaogenic reactionfor arabinose by- nio means est'ablishes that the auxillxvithl Rr xvalue of 0.39 xxvas [AA-arabinose. Further

stu(lv of thlis comlpoun(l is required. Neithier dloesthe negative clhromiiogeniic reactiolns for sutgars xx itllhvdrolysates of ether-insoluble auxin p)roxe that theyxxere not conljugatel xith sugars. Since. negative

r-eactions coukll haxe been (clue to the insufficientamounts of mateirials chroinatogrlhle(l. It is I)pos.i-ble that one of the auxi us among those \\witl Rf,

\ci(l-ether

Netitral-ether

Aqueous

0.16

476 PLAN\T PHYXSIOLOGYE



477SRIVASTAVA-AUXINS FROM IMMATURE CORN KERNELS

values of 0.47, 0.63, and 0.75 was IAA-glucose whichhas been reported by Klambt (11) and Zenk (25)to be formed during the metabolism of IAA. Whenaqueous fraction from cold ethanol extract of im-mature corn kernels was chromatographed in sol-vents used by Klambt and Zenk, namely, isopropa-nol: ammonia: water (80: 15: 5); n-butanol: formicacid: water (10: 1: 2); n-butanol: acetic acid: water(5: 1: 2.2) positive color reactions with Ehrlich andSalkowski reagents were produced approximately atthe same Rf values as reported for IAA-glucose bythe above authors. Since authentic IAA-glucosewas not available for comparison positive proof forits presence in corn cannot be made.

Comnparison of Cold-Ethanol and Ether-EthanolExtractions. Essentially the same compounds weredetected when chromatograms of acid-neutral etherand aqueous fractions of cold ethanol and ether-ethanol extracts from equal amounts of immaturecorn kernels were sprayed with Ehrlich and Salkow-ski reagents. As judged by the intensity of spotsproduced the concentration of IAA and of otherether-fraction auxins was greater on ether-ethanolchromatograms than on cold ethanol chromatogramswhereas no significant differences in the concentra-tion of aqueous fraction auxins were noted betweenthe two extraction procedures. These results werealso confirmed by the bioassay of parallel chromato-grams (table II). In one experiment the IAA re-gion of the chromatogram from ether-ethanol extrac-tion was eluted with ether and the ultraviolet spec-trum of the eluate determinecl. The spectrum ofthe eluate (A.mn 245 mnu, Amax 280 mu, Shoulder 290imi) was very similar to that of IAA indicating thatthe auxin in question was indeed IAA.

Table IIThe Com11parison of Concentrations of Various GrowthSubstances in the Acid-Neutral Ether anid Aqueous

Fraction of Cold Ethanol & Ether-EthanolExtracts of Imnmnatuire Corn Kernels

Mean* conc**mg equiv

Fraction Rf Zone IAA/kg fr wt

Cold Ether-ethanol ethanol

Acid-neutral-ether0.0-0.4 0.04 0.110.5-0.6*** 6.20 25.000.7-1.0 0.09 0.34

Total 6.33 25.45Aqueous

0.0-0.1 0.27 0.330.2-0.5 72.50 76.500.6-1.0 3.07 1.50

Total 75.84 78.33* Mean of two determinations.

** Concentrations determined by Avena bioassay froma IAA standard curve.Zone of IAA.

DiscussionEssentially the same compounds were detected in

ether and aqueous fractions whether the kernels wereextracted with cold ethanol or ether-ethanol. IAAwas positively identified, but no evidence for thepresence of IPyA, TNH,, or IAN was obtainedwith the methods used in the present study.

The auxins of the aqueous fraction described inthis paper gave positive indole reactions, had indolelike ultraviolet spectra, and produced IAA on alkalinehydrolysis. Therefore, they were probably conju-gates of IAA. Though evidence of liberation ofsugars on hydrolysis for all aqueous fractions con-taining auxins was not obtained it is tempting tosuggest that they may represent a group of IAA-sugar conjugates to which IAA-glucose and IAA-arabinose are examples. These compounds are ofwide occurrence in plants (4, 18, 21) and are alsoformed on incubating plant tissues with IAA (11,20, 25) but escape detection with chromogenic re-agents due to their low concentration.

Farrar (5) has recently reported the occurrenceof several ether-insoluble auxins from immaturecorn kernels which were positive to Ehrlich andSalkowski reagents, but negative to ninhydrin andaniline phthalate and decomposed to give IAA inammoniacal chromatography. The ether-insolubleauxins described here resemble those of Farrar intheir chromogenic reactions, but they did not de-compose to any great extent in ammoniacal chroma-tography. In spite of some differences due to dif-ferent procedures of analysis it seems fairly certainthat the same compounds are involved.

Since ether-ethanol extracted greater amounts ofIAA whereas there were no significant differencesin the concentration of the auxins of the aqueousfraction as conmpared to cold ethanol extraction(table II) the increased amount of IAA was not ob-tained at the expense of water soluble auxins, butfrom some different source. Aqueous fractionauxins are, therefore, different from free auxins aswell as bound or reserve forms of IAA, which is inagreement with the argument of Farrar (5). Theauxins of the aqueous fraction may in fact representthe active forms of IAA in plants performing specificfunctions in the growth and development of plants.

SummaryFree and bound auxins of immature corn kernels

were extracted with cold ethanol (-10° overnight)and ether-ethanol (ether 48 hours at 250 followed byextraction of residue with 90 % cold ethanol) re-spectively and the acid-ether, neutral-ether, andaqueous (remaining after ether extractions) fractionswere examined by paper chromatography. Essen-tially the same compounds were detected by chromo-genic reagents and Avena growth test by both ex-traction procedures. No significant differences inthe concentration of aqueous fraction auxins betweenthe two extraction procedures was found. There-



I'LANT P'HXYSIOLOGY

fore, the increased amount of inldoleacetic acid ex-tracte(i l)v ether-ethanol as comp)are(l to cold ethanolextractioni wvas niot release(l fromii the a(lueous frac-tion auxinls hut froIul somile (lifferenlt souirce.

In tlle acid-ether fracti-on ini(loleacetic wx-as idleinti-fied by its activity in the A\-elna cylinder test, chro-mogenic reactionis, and ultraviolet spectrum. Severalother comiipouil(Is that xwere positix e to clhrolmiogeniicreagents and(I active in Av-elna test xx-ere (letecte(l inacid-ether a-lnd neutral-ether fractionis but they x-ereiot i(lenitifie(l.

In the a(queouis fraction- six comllpotund(Is th,at were

positive t) Ehrlich and Salkowxski re.agents and(I x-erealso active in the Avena test wxere dletectedl. Thlesecomlpounlds sllowed indldole-tvl)e ultraviolet spectraand gave rise onl alkalline 1-hydrolysis to ind(lole-aceticacidl, wh-liclh xas i(lentifie(d by color reactions and(iultraviolet spectruml. No evide-lce for the produc-tion of amiiino aci(ls onl lhdrolysis of aqueous fractionauxins was obtained. The release of arabilnose on

hydrolysis as ind(licate(d for onlyl onie of the comii-

poun(ls.

It is stg-estedl that tile aUxin1s of the aqueousfractioll of corin kerniiels may represenit a g-rouil) ofindoleacetic aci(l-su-ar colnjuglteS that are nlot re-

serve or houdlid forms of auxin hut mlay in fact repre-sent the actixe forms of auxin in l)lalts.

Acknowledgment

Preliminary wx ork on this subject xx as carrie(l out atthe Departmiienit of Biology, University of Saskat chexxwan,Saskatooin d(urinig the tenture of a Researclh Fellow ship.The initerest andcl encouragement of Dr. -M. Slhaxw aregratefully ackuno ledoged.

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ether-soluble & ether-insoluble auxins from immature corn ... · ether-soluble &...

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