clhlor - Plant physiology · KAY AND PHINNEY-PLASTID PIGMENT CHANGES ment synthesis in bean and corn. Plant Physiol. 15. ZSCHEILE, F. P. and COMAR, C. L. Influence of pre- 28: 1-14

KAY AND PHINNEY-PLASTID PIGMENT CHANGES

ment synthesis in bean and corn. Plant Physiol. 15. ZSCHEILE, F. P. and COMAR, C. L. Influence of pre-28: 1-14. 1953. parative procedure on the purity of clhlorophyll

14. ZECHMEISTER, L. and CHOLNOKY, L. Principles and components as shown by absorption spectra. Bot.Practice of Chromatography. Pp. 1-362. John Gaz. 102: 463-481. 1941.Wiley and Sons, New York 1948.

STUDIES ON 3-INDOLEACETIC ACID METABOLISM. II. SOME PRODUCTS

OF THE Mi1ETABOLISM OF EXOGENOUS INDOLEACETICACID IN PLANT TISSUES1,2

NORMAN E. GOOD, W. A. ANDREAE AND M. W. H. VAN YSSELSTEINSCIENCE SERVICE LABORATORY, UNIVERSITY SUB POST OFFICE, LONDON, ONTARIO, CANADA

It has long been known that indoleacetic acid(IAA) is taken up by plants and translocated to vari-ous tissues, causing characteristic modifications in thepattern of growth. However the fate of IAA in vivohas received little attention. This is surprising in thatIAA and its derivatives are virtually the only mate-rials of known chemical constitution which both ex-hibit the formative effects of plant growth substancesand occur naturally in plants. The fact that the con-centrations of endogenous IAA are extremely low isno doubt in part responsible for the neglect of an im-portant field of research. The authors of this paperhave attempted to overcome the difficulty by supply-ing much larger amounts from an ambient solution.They are quite aware, however, that the reactionsassociated with relatively high concentrations of ap-plied IAA may differ significantly from metabolicreactions as they occur in nature.

Exogenous IAA participates in several biologicalreactions. Plants are known to contain IAA oxidizingenzymes and part of the IAA administered to plantspresumably undergoes oxidative degradation to prod-ducts which are no longer growth active (8). Hem-berg (3) has noted a rise in "bound auxin" in IAAtreated corn kernels. Siegel and Galston (6) havereported that IAA administered to excised pea rootsbecomes attached to proteins. On the other hand wehave recently found that IAA administered to peastems is conjugated with aspartic acid (1). The pres-ent communication constitutes a report on the exten-tion of our investigations to other plant species.

MATERIALS AND MIETHODS

A sturvey was carried out using 12 plant speciesbelonging to 8 families. The following tissues wereinvestigated: etiolated coleoptiles of oats, corn andbarley all harvested just before the emergence of theprimary leaf; etiolated epicotyls of peas 7 days old;etiolated hypocotyls of sunflower, cucumber and buck-wheat, 7, 4, and 4 days old respectively; etiolatedpotato sprouts 3 to 5 inches long; stems and petiolesof greenhouse grown pea, tomato, and cabbage plants.Immediately after harvesting, the tissue was weighed

1 Received October 11, 1955.2 Contijbution No. 62, Canada Department of Agri-

culture, Science Service Laboratory, University Sub PostOffice, London, Ontario, Canada.

and cut into pieces aproximately 2 inches long. Toeach 100 gms of tissue were added 2 liters of M/60sodium dihydrogen phosphate with or without 60 mgof IAA.3 The floating tissue was incubated at 240 Cfor 24 hours in the dark with gentle shaking. Afterthis period the tissue was removed, thoroughly washed,and frozen at -8° until it could be conveniently ex-tracted. The residual IAA in the ambient solutionswas measured by the acid-ferric chloride (Salkowski)method (8) and in some cases the solutions were etherextracted and chromatographically analyzed for otherSalkowski-positive substances.

To each 150 gm of frozen tissue was added 65 mlof 0.3 N sodium bicarbonate solution. The tissue wasthen ground in a Waring blendor and filtered throughcheese cloth. The resulting brei was saturated withammonium sulfate, infusorial earth was added and theprecipitated proteins were removed by filtration on aBuchner funnel. (The unwashed precipitate from peacontained from 10 to 20 % of the Salkowski-positivematerial. However, when the precipitate was thor-oughly washed by redissolving it in water and repre-cipitating it with ammonium sulphate, practically allthe Salkowski-positive material was in solution. Sincethe washings contained the same substances as thefiltrate and in about the same proportions only thefirst filtrates were regularly investigated in this sur-vey.) The filtrate (pH about 7.5) was extracted oncewith 100 ml and twice with 50 ml of peroxide-freeether, acidified to pH 2.5 with phosphoric acid andextracted with ether as before. Then the acidifiedfiltrate was extracted with 100, 50 and 20 ml portionsof 1-butanol. The aqueous residue was by this timeSalkowski-negative. The acidic extracts were madeslightly alkaline either with sodium bicarbonate solu-tion or with dilute ammonia and taken to dryness, the

3 Some samples of indoleacetic acid from commercialsources are very impure and contain appreciable amountsof indoleacetamide and other Salkowski reactive sub-stances. The following purification procedure, however,has been found to be effective: Commercial IAA is dis-solved in water (about 100 ml/gm) containing an excess

of sodium bicarbonate. The aqueous solution is ex-

tracted several times with 1-butanol, then several timeswith ether. The dissolved ether is removed by aerationand the solution is slowly acidified to pH 3.0 with phos-phoric acid. About 70 %o of the original IAA separateson standing as white, flake-like crystals.

231

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PLANT PHYSIOLOGY

Cs

lkase

t, 11 t.- ,rS

tC-4

.........

A

_..........

-Sdord...i.......

9an.

5....

).

4

0(i

.^, -3w-_ ......

.li _ffix. a-- -_111F .2_I_S_@ x _..:w.tal. ..._wllgO

* ^ :::*_ i_.,i,S .> N as_

._.._ _Fi_...*S_0_|. _,_ .. _r ................ r _.

..E

n

_.. g. .*. .0 w.__.

._

. .-

h

FIG. 1. Typical chriomatograms of extractstreated (T) and untreated (C) plant tissues (sLhypocotyls) obtained by successive alkaline-etheether and acid-butanol extractions. East spot relthe extract from 3 gm fresh wt of tissues. Chgram (A) was developed with an isopropanol-amiacetate-acetic acid solvent and sprayed with thereagent (p-dimethylaminobenzaldehyde-HCI). Ctograph (B) was developed with an isopropanolnia solv-ent and sprayed with the EhrlichChromatogram (C) was developed with the sa

ether extracts in an air stream and the butanol ex-A *t tracts by distillation at reduced pressure. The sol-

vent-free residues were taken up in 1.0 ml of diluteaqueous ammonia or bicarbonate solution. An aliquot(20 ,ul) was immediately chromatographed on paperand the remainder was stored at - 80 C.

The concentrated extracts were chromatographedon 15" x 15" sheets of Whatman #1 filter paper for16 hours at, 240 C, using the ascending solvent tech-niques. The principal partitioning solvents were:

(A) 80 parts of 2-propanol to 20 parts of 8 Nammonium hydroxide solution (v/v).

(B) 80 parts of 2-propanol containing 1.25 %acetic acid to 20 parts of a 20 % aqueous solution ofammonium acetate (v/v).

Equivalent extracts from the untreated and theIAA treated tissues were chromatographed as ad-

!jacent spots. The dried chromatograms were sprayedeither with a modified Salkowski reagent (0.05 MIFeCl3 in 35 % HCl04) or with the Ehrlich reagent

B (1 % p-dimethylaminobenzaldehyde in 50 % alcohol-50 % concentrated HCl). See figure 1. In some casesregions on unsprayed chromatograms correspondingto the colored regions on sprayed chromatograms were

A eluted with water or dilute aqueous ammonia and re-* chromatographed. The resulting purified material

w., swas again eluted, its ultraviolet absorption spectrumdetermined and its hydrolysis products tested forIAA, amino acids and other substances. When thesurvey was completed the frozen acid-ether and buta-nol extracts were thawed andl chromatographed simul-taneouslv (see fig 2).

RESULTSThe Salkowski and Ehrlich-reactive IAA deriva-

tives were similar in the various plants grown underC different conditions. Figure 1 shows typical chroma-

tograms of successive extracts, in this case of sun-flower hypocotyls. Similar chromatograms were madewith extracts from each species. Tissues which ha(lnot been treated with IAA (C) were dev'oid of Sal-

t kowski-reactive material and the only Ehrlich-positivesubstance, which occurred alike in treated and un-treated plants, was tryptophan (spot 5). This wastrue of all 12 plants examined. For purposes of com-parison the extracts of the 12 plant species werechromatographed together to form the composite pic-ture shown in figure 2; space limitation did not permitinclusion of the controls. There were 4 major Sal-kowski-reactive substances and only these have been

of IAA studied in detail. A description of the numerousunflower minor components will entail a, more intensive studyar, acid- of the tissue extracts.presents 3-INDOLEACETAMIDE: All the extracts of IAAiromato-moniuimEhrlichThroma-I-ammo-reagent.me iso-

propanol-ammonia solvent but sprayed with the Salkow-ski reagent (ferric chloride-perchloric acid). Spot 1 issynthetic indoleacetamide, spot 2 is indoleacetic acid andspot 3 is synthetic indoleacetylaspartic acid, each 0.05micromoles. Spot 4 is an unknown acidic substance de-scribed in the text, and spot 5 is tryptophaln.

232



GOOD ET AL-METABOLISM OF EXOGENOUS IAA

treatedl plants, with the posber, contained netutral rapidmatographicallv idlentical wit1, figs 1 and 2). These ma

I

rn

rn~

,..............

*m _

:

4

co

^uXb.$U

co,

W)

........,,

FIG. 2. Chromatograms ofbutanol (B) extracts from the

plant species. Each spot reprgm fresh wt of tissue. The chopedl with an isoplropanol-ammwith the Ehirlieh rieagent. Spacetamide, spot 2 is indoleacetthetic indoleacetylaspartic acid

sible exception of eucum- doleacetamide, gave the purple color typical of IAA-moving substances chro- with the Ehrlich reagent and a slowly developingh 3-indoleacetamide (spot weak color with the Salkowski reagent. A sampleterials, like synthetic in- from corn coleoptiles had the ultraviolet absorption

spectrum of indole compounds with maxima at 280and 288 mu. It yielded IAA and ammonia (as deter-

* mined by the Nessler reaction) in approximately equi-0. xmolar amounts on hydrolysis. Apparently indole-

acetamide occurs in particularly large amounts inIAA treated grasses (fig 2).

3-INDOLEACETIC ACID: Each extract of IAA treatedplants contained an acidic substance chromatographi-cally identical with IAA (spot 2, figs 1 and 2).

Af; _3-INDOLEACETYLASPARTIC AcID: The acid-ether and

butanol extracts of all the IAA treated plants con-*~*tained a slow-moving acidic substance chromato-

graphically identical with the indoleacetylaspartic acid(spot 3, figs 1 and 2) previously reported in pea (1).The earlier report stressed the facts that indoleacetyl-aspartic acid was not extracted by ether and that itdid not occur in barley. Both of these assertions must

* * be qualified. Because of its increased acid strength,N ~ presumably due to the presence of an acylated amino

group adjacent to one of its carboxyls, indoleacetyl-aspartic acid is only extracted by ether from strongly

* acidic solutions (about pH 3.0) and thus can be sepa-rated easily from IAA. Moreover indoleacetylaspartic

0 * acid or some very similar substance does occur in

barley but in such small amounts that its presencewas missed until highly concentrated extracts wereexamined (fig 2).

AN UNIDENTIFIED ACIDIC SUBSTANCE: Anotheracidic material occurred in the acid-ether extracts ofmany IAA treated plants, particularly in the extractsof sunflower (spot 4, fig 1 C), tomato, bean, cucumberand pea. This substance appeared approximatelymidway between IAA and indoleacetylaspartic acidon chromatograms developed with the 2-propanol-ammonia solvent. There was some evidence that twocompounds may have been involved since the Rf valueof the material from sunflower was considerably higherthan that of the comparable material from cucumber.The substance (or substances) gave an immediatebright pink or scarlet color with either acid-ferricchloride (Salkowski) or acid-nitrite sprays and couldlbe readily distinguished from IAA by the speed of thecolor development. It gave a very weak, atypical(grey) color with the Ehrlich reagent. Since none ofit was extracted by ether from weakly basic or neutral

Aor.. soluitions, it is probably a stronger acid or its salts are

less soluble in ether than is the case with IAA. Theundlissociated form, however, was almost quantita-tively removed by ether from acidified aqueous solu-

acid-ether (E) and acid- tions. A sample obtained from sunflower hypocotylsIAA treated tissues of 12 was tunstable to boiling with either acid or base butesents the extiact from 3 no IAA or other Salkowski-positive material could beiromatograms were devel- found after the treatment. The substance could notionia solvent and sprayed be detected among the products of IAA oxidationot 1 is synthetic indole- when the oxidation was brought about enzymaticallyic acid, and spot 3 is syn- by oxygen or non-enzvmatically by perbenzoic acid,1, each 0.05 micromoles. hvpochlorite or permanganate.

233

a:

-.4



PLANT PHYSIOLOGY

None of the compounds (lescribed above appearedin the ambient solution during the incubation of theplant tissues and therefore it is unlikely that the me-tabolism of inadvertently introduced bacteria couldhave contributed any of these derivatives.

Relatively little of the IAA lost from solutioncould be found in the tissue extracts by means of thecolor tests used. Table I shows the quantitative dis-crepancy between the disappearance of IAA and theaccumulation of extractable Salkowski-reactive mate-rial in the plants. An attempt to identify the missingunreactive substances has been made, witlhout success

TABLE ITHE CONVERSION OF IAA BY PLANT TISSUES INTO

SUB.STANCES LESS CHROMOGENIC WITHTHE SALKOWSKI REAGENT

SALKOWSKI DECREASE INCOLOR ** SALKOWSKIDEVELOPED LOSS OF REACTION OF

WITH COMBINED IAA THE AMBIENTTISSUE TISSUE EXTRACTS FROM SOLUTION

AND EXPRESSED THE ACCOUNTEDAS AMOUNT OF AMBIENT FOR BY THEIAA REQUIRED SOLUTION REACTION OFTO GIVE THE THE TISSUESAME COLOR EXTRACTS

Dig mg /oCorn ........ 6 52 11.5Barley 6 64 9.5Oats* 2.5 54 4.5Onion 5 50 10Pea 12.5 70 18Bean 7 20 3.5Tomato 2.5 15 16.5Potato 4 34 12Cabbage 2.5 0 ( ?)Cuctumber 1 21 5.0Sunflower 3 15 20Buckwheat .. 4 16 25

The data refer to 150 gm fresh wt of tissue incubatedfor 24 hrs at 240 C in 3 liters of M/60 NaH2PO4 solutioncontaining 90 mg IAA.

* 75 gm fresh wt.** IAA in the ambient solution and Salkowski reactive

materials in the tissue extracts were determined by themethod of Tang and Bonner (8) except that the read-ings were taken 2 hrs after the addition of the Salkowskireagenit (1) .

to (late. Alkaline hydrolysis of the tissue proteins didnot release IAA or recognizable degradation products.Sprays containing nitrous acid, diazotized sulphanilicacid, diazotized p-nitroaniline, and 2,4-dinitrophenyl-hydrazine did not reveal significant amounts of newsubstances on the chromatograms of tissue extracts.Similarly, unspecific oxidants such as ferric chloride-ferricyanide mixtures (letected only the spots alreadyobserved with the aid of the conventional indole re-agents. However, the precipitated proteins, the aque-oUs residue after butanol extraction and the ambientsolution have not vet been examined for Salkowski-and Ehrlich-negative IAA derivatives. It, would seemthat the investigation of these more elusive substances

must await the development of improved methods fortheir detection.

DISCUSSIONIt is not known which compoundls derived from

exogenous IAA are also formed from endogenous IAAand consequently the pertinence of our observationsto the problems of the normal metabolism of plantgrowth hormones has not been established. Otherworkers have reported a number of Salkowski-reac-tive substances from plant extracts. Among thesewere indoleacetic acid (2), indolepyruvic acid (7), theethyl ester of indoleacetic acid (5), and indoleacetoni-trile (4). Indoleacetylaspartic acid and indoleaceta-mide have not yet been reported as naturally occur-ring and judging by the published information thereis no reason to believe that they have been encoun-tered without being recognized.

In spite of the large number of Salkowski-positivesubstances found in the treated tissuies (at least 7 insunflower) most of the IAA lost from the ambientsolution was converted into Salkowski- and Ehrlich-negative substances, or at least, into substances whichgive very much weaker colors with both these re-agents. Since the Ehrlich reagent as used in the ex-periments reacts fairly generally with 3-substittutedindoles, it is probable that the dcegradation involvesan early ring cleavage. Radioactive indoleacetic atcidwith C14 in the aromatic nucleus is being synthesizedand will be employed in future studies to locate theseless easily recognized substances.

SUMMARYTissues from twelve plant species have been ircu-

bated with indoleacetic acid. Extracts of these tisstueshave been examined for IAA derivatives.

A large number of substances which give charac-teristic colors with the Salkowski or Ehrlich reagentswere found in all the IAA treated tissues. Thoseoccurring most generally and in largest amounts w-ere,on the basis of chromatographic behavior and hy-drolysis products, indoleacetamide, unchanged indole-acetic acid, and indoleacetylaspartic acid. An un-known acidic compound which reacts with the Sal-kowski reagent but not with the Ehrlich reagentoccurred in several plants.

No convincing qualitative (lifferences in the lprod-uicts of IAA metabolism were noted among the plantsinvestigated. However, indoleacetamide predlominatedin the grasses and indoleacetylaspartic acid l)redoomi-nated in the legumes and in onion.

MWost of the indoleacetic acid which disappearedcouldk not be accounted for by substances reactingwith either of the reagents.

LITERATURE CITED

1. ANDREAE, W. A. and GOOD, NORMAN E. The forma-tion of indoleacetylaspartic acid in pea seedlings.Plant Physiol. 30: 380-382. 1955.

2. BENNET-CLARKE, T. A., TAMBIAH M. S. and KEFFORD,N. P. Estimavtion of plant growth substances by

234



GOOD ET AL-METABOLISM OF EXOGENOUS IAA

partition chromatography. Natur e 169: 452453.1952.

3. HEMBERG, T. Studies on the balance between freeandl bound auxin in germinating maize. Physiol.Plantarum 8: 418-432. 1955.

4. JONES. E. R. H., HERBERT, H. B., SMITH, G. F. andBENTLEY, J. A. 3-Indolylacetonitrile: a naturallyoccurring plant growth hormone. Nature 169:485-487. 19.53.

5. REDEMANN, S. T., VT 1TTWER, S. H. and SELL, H. M.The fluit-setting factor from the ethanol extracts

of immature corn kernels. Arch. Biochem. Bio-phys. 32: 80-84. 1951.

6. SIEGEL, M. G. and GALSTON, A. W. Experimentalcoupling of indoleacetic acid to pea root proteinin vivo and in vitro. Proc. Nat. Acad. Sci., U. S.39: 1111-1118. 1953.

7. STOWE, B. B. and THIMANN, K. V. Indolepyruvicacid in maize. Nature 172: 764. 1953.

8. TANG, Y. W. and BONNER, J. The enzymatic inacti--vation of indoleacetic acid. I. Some characteristicsof the enzyme contained in pea seedlings. Arch.Biochem. 13: 11-25. 1947.

STUDIES ON 3-INDOLEACETIC ACID TMETABOLISMI. III. THE UPTAKE OF3-INDOLEACETIC ACID BY PEA EPICOTYLS AND ITS CONVERSION

TO 3-INDOLEACETYLASPARTIC ACID 1 2

W. A. ANDREAE AND M. W. H. VAN YSSELSTEINSCIENCE SERVICE LABORATORY, CANADA DEPARTMENT OF AGRICULTURE

UNIVERSITY SUB POST OFFICE, LONDON, ONTARIO

In two earlier publications (2, 5) it was reportedthat 3-indoleacetic acid (IAA) administered to planttissues is taken up and converted in part to deriva-tives which still give positive Salkowski (acid-FeCl3)reactions. The two major derivatives which reactwith this reagent were identified as 3-indoleacetyl-aspartic acid and 3-indoleacetamide. Indoleacetamidepredominates in grasses while indoleacetylaspartic acidis by far the most abundant Salkowski reactive de-rivative accumulating in legumes.

The present paper deals with factors affecting theuptake of IAA and its conversion to Salkowski reac-tive dlerivatives in pea epicotyls. Paper chromato-graphic studies have shown that indoleacetylasparticacid represents about 90 % of the Salkowski reactiveIAA derivatives and consequently the present studyis essentially one of the formation of indoleacetyl-aspartic acid. In this investigation IAA and indole-acety-laspartic acid were estimated independently byextracting lyophilized tissues with ether; indoleacetyl-aspartic acid, which is insoluble in ether under theseconditions was then extracted with bicarbonate solu-tion, leaving a Salkowski unreactive residue. For thesake of convenience, the Salkowski positive materialsin the ether and bicarbonate fractions will be referredto as "IAA" and "indoleacetylaspartic acid" respec-tivelv although in fact both fractions also containedsmall amounts of unknown IAA derivatives. The pro-cedure used was not sensitive enough to detect theIAA and other Salkowski positive materials whichoccur naturally in the tissues.

MATERIALS AND METHODSPea epicotyl sections were obtained from Alaska

peas growni on vermiculite in the dark at 270 C (800 F)

1 Received January 24. 1956.2 Contribution No. 64, Science Service Laboratory,

Cana(la Agriculture, Lniv-ersity Sub Post Office, London,Ontario.

and 80 % relative humidity, exposed to red liglht for2 hours on the 6th day and harvested on the seventh.The epicotyls, with their terminal buds removed, werecut into approximately 2-inch sections and 10-gramlots were floated on 500 ml of MI/60 NaH2PO4 solu-tion containing varying amounts of IAA. The pH ofthe ambient solution was about 4.6. The suspendedepicotyls were shaken in the dark at room tempera-ture for intervals of varying duration. Before andafter incubation 1-ml aliquots were removed from theambient solution and treated with 4 ml of the Sal-kowski reagent (0.001 M FeCl3 in 14 N H2SO4). After20 minutes the intensity of the pink color was deter-mined photometrically as described by Tang andBonner (9).

After incubation with IAA the tisues were removedfrom the solution and washed with water. In someexperiments they were then immediately frozen andly3ophilized; in other experiments the tissues wereplaced for various intervals of time on glass plates inan atmosphere saturated with water vapor in the darkat room temperature after which they were frozenand lyophilized.

In both cases the lyophilized epicotyls were weighedand ground in a Wiley mill to pass a 40-mesh sieve.Two-hundred-milligram aliquots of the dry powderwere then extracted 4 times with 5 ml of water-satu-rated, peroxide-free ether. The ether extracts werecombined, filtered, evaporated to dryness and taken upin 2 ml of 0.1 N NaHCO3. To 1 ml of the concen-trated extract was added 4 ml of the Salkowski re-agent and the intensity of the color formed deter-mined as described above. The powder, after etherextraction, was suspended in 4 ml of 0.1 N NaHCO3overnight at 50 C and then centrifuged. To 1 ml ofthe supernatant solution were added 4 ml of Sal-kowski reagent and the intensity of the mauve colorcharacteristic of the Salkowski reaction with indole-acetylaspartic acid was determined except that the

23 -



Documents

clhlor - Plant physiology · KAY AND PHINNEY-PLASTID PIGMENT CHANGES ment synthesis in bean and corn. Plant Physiol. 15. ZSCHEILE, F. P. and COMAR, C. L. Influence of pre- 28: 1-14