Plant Physiol. (1990) 94, 485-4910032-0889/90/94/0485/07/$01 .00/0

Received for publication February 28, 1990Accepted May 8,1990

In Vitro Oxidation of Indoleacetic Acid by Soluble Auxin-Oxidases and Peroxidases from Maize Roots

Roland Beffa1, Hilary V. Martin, and Paul-Emile Pilet*Institute of Plant Biology and Physiology of the University, CH-1015 Lausanne, Switzerland

ABSTRACT

Soluble auxin-oxidases were extracted from Zea mays L. cvLGlI apical root segments and partially separated from peroxi-dases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to amolecular mass of 32.5 kilodaltons and a minor peak at 54.5kilodaltons. Peroxidases were separated into at least four peaks,with molecular masses from 32.5 to 78 kilodaltons. In vitro activityof indoleacetic acid-oxidases was dependent on the presence ofMnCI2 and p-coumaric acid. Compound(s) present in the crudeextract and several synthetic auxin transport inhibitors (including2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhib-ited auxin-oxidase activity, but had no effect on peroxidases. Theproducts resulting from the in vitro enzymatic oxidation of [3H]indoleacetic acid were separated by HPLC and the major metab-olite was found to cochromatograph with indol-3yl-methanol.

Two pathways for the oxidative degradation of IAA havebeen established. The first involves oxidation of the indolenucleus (32) by a specific enzyme which catalyzes the forma-tion of oxindole-3-acetic acid (OxIAA2; 23), whereas thesecond pathway consists of the oxidative decarboxylation ofthe side chain. Peroxidases (EC 1.11.1.7) from numerousplant species have been shown to catalyze this oxidativedecarboxylation (27), leading to the formation of either indol-3yl-methanol (17) or 3-methyleneoxindole (6). The ratio ofthe production of these last two compounds is influenced byseveral factors including the nature of the cofactors added tostudy the reaction in vitro, the pH, and the enzyme/substrateratio (4, 26).The breakdown of IAA catalyzed by peroxidases has been

studied in a number of systems, and the requirement foradded cofactors and for H202 differs between species. Thedegradation of auxin by HRP may occur without addedcofactors, and in the presence or absence of H202 (6). How-ever, tomato peroxidases are dependent on the presence ofH202 for the oxidation of IAA (9) and fail to catalyze thereaction in the presence of compounds such as 2,4-dichloro-

'Present address: Department of Botany. University of Georgia,Athens, GA 30602.

2Abbreviations: OxIAA, oxindole-3-acetic acid; 3,3-DGA, 3,3-dimethylglutaric acid; DIBA, 3,5-diiodo-2-hydroxybenzoic acid;DIHB, 3,5-diiodo-4-hydroxybenzoic acid; DNP-alanine, N-2,4-dini-trophenyl-3-alanine; HRP, horseradish peroxidase; NPA, N-1-na-phthylphthalamic acid; TIBA, 2,3,5-triiodobenzoic acid.

phenol, p-coumaric acid, and manganese (10). Studies ofotherplant peroxidases showed that these compounds were re-quired, separately or together, to catalyze IAA-oxidation inwheat seedlings (34) or that they greatly increased the enzymeactivity in maize leaves (15) and in lentil roots (22).Most attemps to separate the two activities (IAA oxidases

and peroxidases) from plant tissues, especially from horse-radish, have so far been unsuccessful, as both types of activitymoved together during purification (7). Nevertheless, IAA-oxidases were, at least partially, separated from peroxidaseswhen they were purified from tobacco roots (28), from crowngall tissue culture cells (24), or from mung bean cotyledons(30). In this case (30), IAA oxidation was inhibited by H202in contrast to the results cited above (9).

In the present investigation, IAA-oxidases are partially sep-arated from peroxidases of maize (Zea mays L. cv LG11)root apical segments. Different properties of the enzymesincluding their molecular masses are determined and theproduct(s) of the oxidative degradation of IAA are analyzedby reverse phase HPLC.

MATERIALS AND METHODS

Plant Material

Selected caryopses of Zea mays L. cv LG 1 (AssociationSuisse des Selectionneurs, Lausanne) were germinated androots grown as previously described (1) in the dark at 19 +1°C. After 46 ± 2 h, seedlings with 15 ± 3 mm straightprimary roots were selected. Apical segments (10 mm) wereexcised and immediately frozen in liquid nitrogen.

Extraction and Purification of Enzymes

All the steps of the purification procedure were carried outat 4°C. Apical root segments (100 pieces, about 1 g freshweight) were ground in liquid nitrogen to a fine powder,homogenized with 4 mL ammonium acetate (20 mM) bufferat pH 6.8 ± 0.1 (22). The homogenate was centrifuged twice(20,000g, 20 min), and each time the pellet was discarded.The second supenatant was retained as the crude extract andusually 500 IuL were set aside for the determination ofenzymeactivities.

In the first step of purification, the crude extract was passedthrough a Sephadex G-25 (Pharmacia, Sweden) column (2.2x 51 cm) previously equilibrated with ammonium acetatebuffer (flow rate: 18 mL h-'). Fractions were collected every20 min and those corresponding to the protein peak andcontaining both enzyme activities (peroxidases and auxin-

485 www.plantphysiol.orgon April 15, 2020 - Published by Downloaded from

Copyright © 1990 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 94, 1990

oxidases) were pooled and concentrated by ultrafiltration(Diaflo YM 10 membrane, Amicon Co.) to form the 'G-25extract' (final volume: 8-10 mL); 1 mL was kept for thedifferent enzyme analyses.For the second step of purification, the G-25 extract was

concentrated to a final volume of 350 ,uL using a Minicon B15 filter (Amicon Co.) and applied to a column packed witha Biogel P-100 minus 400 mesh gel (Bio-Rad; 25). Fraction-ation was carried out in a glass column (1.5 x 100 cm)previously equilibrated with ammonium acetate buffer (flowrate: 3 mL h-'), and the fractions collected every 20 min wereanalysed to determine the peroxidase and IAA-oxidase activi-ties and the protein content. The total procedure was per-formed four times.

Peroxidase Assay

Peroxidase activity (EC 1.11.1.7) was determined colori-metrically using guaiacol and H202 (Perhydrol; Merck) bothat 5.5 mm in 10 mm 3,3-dimethylglutaric acid (3,3-DGA)-NaOH at pH 6.00 ± 0.05 (5); 100 jlL of extract were testedin a final volume of 3.3 mL. The oxidation of guaiacol wasdetermined by measuring the absorbance at 470 nm after 10min incubation at 30.0 ± 0.5°C. Each value was the mean ofat least four separate experiments, each done in duplicate.

IAA-Oxidase Assay

The standard reaction mixture contained in a final volumeof 1 mL: 6.66 mm phosphate buffer (KH2PO4/Na2HPO4) atpH 6.00 ± 0.05, 100 ,LM MnC12, 50 ,uM p-coumaric acid, 15jig IAA (85.6 AM), and 200 AL of the extract tested. Assayswere conducted at 24.0 ± 0.5°C for 40 or 50 min with shaking(200 rpm). The Salkowski reagent (2 mL), modified as de-scribed previously (20), was then added and the destructionof IAA was determined by measuring the absorbance at 535nm after 30 min. Each value was the mean of at least fourseparate experiments done in duplicate.

Enzyme Effector Assays

The influence of cofactors-manganese (MnCl2) and p-coumaric acid (22)-and auxin transport inhibitors such asTIBA, DIBA, DIHB, or NPA was tested on both enzymeactivities, after different purification steps, using the proce-dure described above.

Protein Assay and Molecular Mass Determination

Protein was determined according to Bradford (3) and BSAwas used as the standard. Apparent protein mol wt weredetermined by gel filtration using the Bio-Gel P-100 minus400 mesh which was found to have an optimal resolvingpower and little risk ofprotein denaturation (25). The solutionof standard proteins used to calibrate the column contained1% (w/v) each of BSA, 66 kD (Sigma); HRP, 44 kD (Fluka);chymotrypsinogen, 25 kD (Merck); hen egg white lysozyme,11 kD (Sigma); dextran blue 2000 (Pharmacia) and DNP-alanine (0.1%, Sigma). The elution of each compound wasfollowed either by their absorbance at 280 nm or by proteinquantitation or by measuring their activity (HRP: see perox-

Table I. Auxin-Oxidase and Peroxidase Activities of Crude andG-25 Extract

Protein is given in meq BSA contained in the total volume of theextract. IAA-oxidase and peroxidase activities are expressed in arbi-trary units. One unit of IAA-oxidase activity is equivalent to 1 tg IAAmetabolized by 1 mL of extract in 40 min; 1 unit of peroxidase activitycorresponds of a AA470 of 1.0 for 1 mL of extract in 10 min. The totalactivity corresponds for both enzymes to their activity in the totalvolume of each extract and the specific activity corresponds to thetotal activity expressed per unit of protein quantity.

Crude Extract G-25 Extract

Volume (mL) 3.77 9.17Protein 8.00 ± 0.42 2.32 ± 0.14IAA-oxidase

Total activity 13.6 ± 9.2 303.5 ± 16.1Specific activity 1.7 130.8

PeroxidaseTotal activity 321.2 ± 14.7 84.4 ± 12.8Specific activity 40.2 36.4

idase assay; lysozyme: see ref. 2) or by immunodetection, forBSA, using a rabbit anti-BSA serum and the Ouchterlonydouble diffusion method (18). The calibration of the columnwas repeated three times and no contaminations were ob-served in the peak fractions containing each protein. Thelinear regression analysis between the log of the mol mass ofthe proteins and the elution volume of the respective peakfraction yielded a correlation coefficient of 0.995.

Identification of IAA Metabolites

For each peak fraction, both IAA-oxidase and peroxidaseactivities were tested using, in the same sample, nonradioac-tive and [5-3H]IAA (specific activity, 25 Ci/mmol; Amersham

A30

- p-coumaric acid- 10 uM*-- 50 uM

20- ;-C lOOuM

30B

20 9

Zi ° LU N~~0 40

0 10 100 0 10 100

Mn,CI2 CONCENTRATION IN ,uM P-COUMARIC ACIDI CONCENTRATION IN ijM

Figure 1. Effects of (A) MnCl2 and (B) p-coumaric acid (MnCI2 con-centration 100 jM) on the in vitro IAA-oxidase activity of the G-25extract. IAA-oxidase activity is expressed in units of IAA metabolized(see Table I). Vertical bars represent SE of 20 different extracts,analyzed in duplicate.

486 BEFFA ET AL.

-1

www.plantphysiol.orgon April 15, 2020 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.

IAA METABOLITES PRODUCED BY SOLUBLE MAIZE ROOT IAA-OXIDASES

305 DIHB

NPBA{NJPA20 J) \

0

10~~ ~ ~~~~IDIBA_*10 1

<

0 0.1 1 10 100 1000CONCENTRATION IN uM

Figure 2. Effects of synthetic auxin transport inhibitors (TIBA, DIBA,DIHB, NPA) on the in vitro IAA-oxidase activity of the G-25 extract.IAA-oxidase activity is expressed in units of IAA metabolized (seeTable I). Data are the mean of 20 different extracts, analyzed induplicate, and the SE is reported.

International plc.) as substrate and the metabolites formedwere analyzed by reverse phase HPLC. To determine theIAA-oxidase activity, the incubation medium (200 ML finalvolume) was the same as that described above except that [5-3H]IAA (36.66 kBq, 0.20 Mm) was added, the final concentra-tion of nonradioactive IAA was 57 Mm, and 40 ML of extractwere used. To analyse peroxidase activity, the medium (200ML final volume) contained, in 3,3-DGA buffer, [5-3H]IAA(36.66 kBq, 0.20 Mm), nonradioactive IAA at 28.5 gM, H202at 0.5 mm, and 40 ML of extract. At the end of the incubationperiod, the samples were diluted 20-fold in the appropriatesolvent used for HPLC analyses and kept on ice.

Analyses by reverse phase HPLC were performed as alreadydescribed ( 14) using two different gradients mentioned in theresults. The identification of the main metabolite of IAA wasdetermined by comparison of the retention time and fluores-

Table II. Effects of TIBA, DIBA, DIHB, and NPA on PeroxidaseActivity of the G-25 Extract

Peroxidase activity is expressed in arbitrary units (see Table I).Each value is the mean of 20 different extracts, analyzed in duplicate,and the SE iS reported.

Treatments Concentration Activity

mM

Control 9.2 ± 0.4+ TIBA 20 9.0 ± 0.5+ DIBA 50 8.7 ± 0.4+ DIHB 50 9.3 ± 0.4+NPA 10 9.2±0.7

cence spectra (excitation and emission) with those of standardcompounds analyzed by the same procedure.

RESULTS

Properties of the G-25 Extract and Comparison with theCrude Extract

The auxin-oxidase and peroxidase activities ofboth extractsare shown in Table I. After gel fractionation on Sephadex G-25 and ultrafiltration, the total peroxidase activity decreasedto the same extent as the protein content and, consequently,the specific activity remained constant. On the other hand, alarge increase ofthe total and specific activity of IAA-oxidasesoccurred, which could be the consequence of the removal ofinhibitory substance(s) present in the crude extract. The na-ture of these compounds has not been checked, but the lossof IAA-oxidase activity by mixing the G-25 extract withseveral fractions which were eluted after the void volume(data not shown) indicates the presence of probably morethan one inhibiting compound having a molecular mass lowerthan 5 kD.The effects of cofactors such as manganese (MnCl2) and p-

coumaric acid on the IAA-oxidase activity (Fig. 1) show thatboth compounds increased the in vitro enzymatic oxidationof IAA in the G-25 extract. No such effects were observedusing the crude extract and, in both cases, peroxidase activitywas not affected by either of these compounds (data notshown).

Auxin-oxidase activity was reduced by several inhibitors ofauxin transport (Fig. 2). The in vitro oxidation of IAA wasstrongly inhibited by TIBA, less by DIBA and NPA, and veryweakly by DIHB (50% inhibition by about 5 ,uM TIBA; 50Mm NPA; 100 Mm DIBA; 500 gM DIHB). Other acidic com-pounds like benzoic acid or abscisic acid, even at high con-centration (100 uM) did not significantly affect the in vitroIAA-oxidase activity (data not shown). Peroxidases were notinhibited by any of these compounds (Table II).

1.00

0.75

00

7WoE<

u~0,<0

-

Ln ILz oLfLWzQL D3

0.50

0.25 _

o40 50 60 70 80

FRACTION NUMBER

20

150

WN*.

100

5<U-0=1LOZ

Figure 3. Elution profile of the size-exclusion chromatography onBio-Gel P-100. Units of IAA-oxidase and peroxidase activity aredefined in Table l; the protein content is expressed in LA595 per mLof extract and the molecular mass corresponding to each peakfraction in kD.

c

-0.1

-0.05

2

487

www.plantphysiol.orgon April 15, 2020 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 94, 1990

80

40

,- O

C)

0

D 40a:IrJ

12 20

LL0ILJ

0LID

40

20

40

B

C IL0 5 10 15 20 25

TIME IN MINUTES

Figure 4. Radioactivity (percentage of total radioactivity) in 0.5 minfractions obtained by HPLC of (A) [5-3H]IAA solution, (B) IAA-oxidasepeak fraction 32.5 kD, and (C) peroxidase peak fraction 78 kD.

Aliquots of the respective peak fractions had been incubated for 50min in medium (pH 6.0) containing MnCI2, p-coumaric acid, nonra-dioactive IAA, and [5-3H]IAA. Elution was at 2 mL/min using a lineargradient of methanol and ammonium acetate buffer (pH 6.0); 0 to75% methanol over 25 min.

Fractionation on Bio-Gel P-100 Gel

The results of analyses of the fractions eluted from the Bio-Gel P-100 are given in Figure 3. IAA-oxidase activity was

separated from the major part of proteins and peroxidases. Inall four experiments the same elution profile appeared andfractions containing a high IAA-oxidase activity showed a lowperoxidase activity and vice versa. The molecular mass of themost active IAA-oxidase was 32.5 kD and that of peroxidasesvaried between 32.5 and 78 kD. It must be pointed out thatthe small IAA-oxidase peak observed at 54.5 kD was alwayseluted one fraction later than the peroxidase peak at 56.5 kD.

Identification of the IAA Metabolites

For each peak fraction, both IAA-oxidase and peroxidaseactivities were tested with nonradioactive IAA and [5-3H]IAAas substrate in the same sample. The metabolites formed wereanalyzed by reverse phase HPLC using two solvent systems(Figs. 4-6). Since results for the three peroxidase peaks (78,56.5, and 44 kD) were similar, only the data obtained for the78 kD peak are shown.

When IAA was incubated for 50 min with MnC12, p-

coumaric acid, and an aliquot ofthe fraction tested, the HPLCanalyses using the first solvent system (buffer pH 6.0/MeOH,Fig. 4) showed that the 32.5 kD fraction was the most activeand two IAA-metabolites were radioactively marked. Com-parisons of the retention time, measured by fluorescence, ofseveral standard compounds with that of IAA-metabolites(Table III) led to the conclusion that the main product formedby the 'IAA-oxidase' fraction (32.5 kD) could be indol-3yl-methanol. On the other hand, the incubation of the same

fractions with IAA and H202 did not lead to the oxidation ofIAA (Fig. 5).

Similar results were observed when a second solvent system(buffer pH 5.5/acetonitrile) was used for the HPLC analyses.Incubation of IAA with the cofactors and an aliquot of thefraction tested led to the formation of one main compound(Fig. 6), and the 32.5 kD fraction was the most active. Nodegradation product appeared when IAA was incubated withH202 and the respective enzyme extracts (data not shown).Comparisons of the retention time of the IAA-metabolites,determined using the fluorescence signal, with that ofstandardcompounds (Table III) confirm that the major product formedis indol-3yl-methanol. Moreover, similar fluorescence spectra(excitation and emission) were found for both the majormetabolite formed and indol-3yl-methanol (Fig.7).

DISCUSSION

Manganese and p-coumaric acid were found to increasegreatly the in vitro IAA-oxidase activity but only after theremoval of low molecular mass inhibitor(s) (<5 kD) from the

80

e 400

0

a:

L

g 60

z 40v

UJ 20

0 5 10 15 20 25

TIME IN MINUTES

Figure 5. Radioactivity (percentage of total radioactivity) in 0.5 minfractions obtained by HPLC of peak fractions 32.5 kD (A) and 78 kD(B), both had been incubated for 50 min in medium (pH 6.0) containingH202, nonradioactive IAA and [5-3H]IAA. Elution was at 2 mL/minusing a linear gradient of methanol and ammonium acetate buffer (pH6.0); 0 to 75% methanol over 25 min.

I

A , _ _ ,

,*00*-

I

I |'

' L

II I ," ft -I

A

S--.. %- so---------------j

488 BEFFA ET AL.

www.plantphysiol.orgon April 15, 2020 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.

IAA METABOLITES PRODUCED BY SOLUBLE MAIZE ROOT IAA-OXIDASES

>.

u

>

0

U-

0

w 40-J

4

o 20

z

40

20

B Ij

C

0 5 10 15 20 25

TIME IN MINUTES

Figure 6. Radioactivity (percentage of total radioactivity) in 0.5 minfractions obtained by HPLC of (A) [5-3H]IAA solution, (B) IAA-oxidasepeak fraction 32.5 kD, and (C) peroxidase peak fraction 78 kD.

Aliquots of the respective peak fraction had been incubated for 50min in medium (pH 6.0) containing MnCI2, p-coumaric acid, nonra-dioactive and [5-3H]IAA. Elution was at a rate of 2 mL/min using alinear gradient of acetonitrile and ammonium acetate buffer (pH 5.5);15 to 10% acetonitrile over 10 min and then isocratic at 10%acetonitrile over 15 min.

crude extract. Studies using other plant material showed thatthese cofactors may be required, separately or together, tocatalyze the in vitro oxidation of IAA (15, 22, 34).

In the first step of purification (Sephadex G-25), total andspecific activities of IAA-oxidases increased after the separa-tion of low molecular weight compounds from the proteinfraction, whereas the specific activity of peroxidases did notchange. This suggests the presence of endogenous com-

pound(s) which do not affect peroxidase activity, but stronglyinhibit the activity of IAA-oxidases extracted from maizeroots, as in lentil roots (22) or pea epicotyls (31).

Synthetic inhibitors ofauxin transport such as TIBA, DIBA,DIHB, and NPA which decrease the efflux of [3H]IAA frommaize roots (12, 13) inhibited IAA-oxidase activity. Triiodob-

enzoic acid is the most active compound tested. When theiodine in position 2 was replaced by a hydroxyl group, whichis smaller and less electrophilic (DIBA), a higher concentra-tion was necessary to obtain the same inhibition. When theOH group was moved from the position 2 to the position 4of the benzoic acid molecule (DIHB), the inhibitory effect ofthe compound became very weak.The dual role of these synthetic substances as inhibitors of

auxin transport and as inhibitors of IAA oxidase leads us tomake two comments. The first concerns the mode of actionof synthetic inhibitors of auxin transport. These substancesprovoke various physiological effects such as the inhibition ofroot growth and gravireaction (1, 13), changes in the morpho-logical polarity of tissue cultures (19), and the stimulation ofcoleoptile growth (33). These phenomena may be partly at-tributable to IAA accumulation (33) caused by the inhibitionof IAA efflux from the tissue (12, 13). However, we suggestthat IAA accumulation due to the inhibition of IAA metab-olism should not be ruled out. The second remark concernsthe nature of endogenous inhibitors of IAA oxidase. Wesuggest that these subtances might also act as endogenousinhibitors of auxin transport. In this context it is interestingto note that certain flavonoids, including kaempferol, havelong been known to inhibit IAA oxidase (16), and that similarsubstances, again including kaempferol, have recently beenshown to perturb the transport of auxin at the NPA-sensitivesite (8).

After the second step of purification (Bio-Gel P-100) themain auxin-oxidases were separated from the main peroxi-dases. Auxin-oxidases were resolved into one main peakcorresponding to a molecular mass of 32.5 kD, close to thatfound in lentil roots (20), and another minor peak at 54.5kD. Peroxidases were separated into at least four peaks, withmolecular masses from 32.5 to 78 kD. This is similar to theresult reported for wheat leaves (25-85 kD; 34). It should alsobe mentioned that when different tissues of maize roots(cortex and stele, for example) were separated before theextraction and purification of both enzyme systems, a betterseparation was obtained (R Beffa, P-E Pilet, unpublisheddata).

Reverse phase HPLC analyses of the metabolites resultingfrom the in vitro oxidation of IAA showed that indol-3yl-methanol was the major metabolite produced when IAA wasincubated with aliquots of the 32.5 kD peak fraction which

Table l1l. Retention Times of Standard Compounds and the MajorMetabolite Formed by IAA-Oxidases of the 32.5 kD Peak Fraction

Retention time (in min) was measured by fluorescence (280/350nm). HPLC elution was done either with buffer pH 6.0/methanol (seeFig. 4) or with buffer pH 5.5/acetonitrile (see Fig. 6). lndol-3yl-carboxylic acid was eluted later (1.7 min) than indol-3yl-methanol inthe first solvent system (data not shown) and indol-3yl-aldehyde hadno fluorescence under these conditions. Each analysis was done atleast 4 times.

Compounds pH 6.0/MeOH pH 5.5/MeCN

lndol-3yl-acetic acid 9.93 4.51lndol-3yl-methanol 14.68 9.90Major metabolite formed 14.59 9.97

489

www.plantphysiol.orgon April 15, 2020 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 94, 1990

A

265.9

250 300 350 400x of excitation in nm

C

286.7I354. 3

250 300 350 400X. of emission in nm

265.9

250 300 350 400-A. of excitation in nm

0

288.5 I 3543

250 300 350 400X. of emission in nm

Figure 7. Fluorescence spectra of (A, C) stand-ard indol-3yl-methanol and (B, D) the major prod-uct formed by IM-oxidase activity contained inpeak fraction 32.5 kD. The metabolite was col-lected after elution with the solvent system bufferpH 6.0/MeOH and excitation (B) and emission(D) spectra were done immediately.

contained the major IAA-oxidase activity. Maize root perox-idases, even with H202 as cosubstrate, did not oxidize IAA(at least under these conditions) unlike HRP (17). Indol-3yl-methanol has been found in several species including pea ( 11)and scots pine (29). Moreover, time-course analyses of the invitro oxidation of auxin showed that indol-3yl-methanol isthe first product formed and cell wall bound auxin-oxidases,also purified from maize roots, metabolized IAA to indol-3yl-methanol (HV Martin, R Beffa, P-E Pilet, unpublished data).Thus in maize, the oxidative degradation of IAA can bedivided into at least two pathways: the oxidation ofthe indolenucleus by a specific enzyme which catalyzes the formationof OxIAA (23) and the oxidative decarboxylation of the sidechain by the auxin-oxidases which leads to the production ofindol-3yl-methanol.The results presented here show that the main auxin-

oxidases involved in the oxidative decarboxylation of IAAcan be separated from peroxidases and lead, in vitro, to theformation of indol-3yl-methanol. Moreover, compound(s)present in the crude extract as well as synthetic inhibitors ofauxin transport, inhibit auxin-oxidases without having anyeffect on peroxidases. The existence of a specific enzymaticsystem which catalyzes the oxidative degradation ofIAA mayhave an important role in regard to the fine regulation of thecellular level of auxin which appears to play a significant partin the control of growth (21).

ACKNOWLEDGMENTS

The authors wish to thank Dr. P. Bernasconi for his helpfulsuggestions concerning the determination of the molecularweights of the enzymes, and Mrs. M. Wirth for voluntaryassistance with the HPLC analyses.

LITERATURE CITED

1. Beffa 1, Martin HV, Pilet P-E (1987) A comparison between3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoicacid. I. Effects on growth and gravireaction of maize roots.Physiol Plant 71: 30-36

2. Bernasconi P, Locher R, Pilet P-E, Jolles J, Jollis P (1987)

Purification and N-terminal amino-acid sequence of a basiclysozyme from Parthenocissus quinquifolia cultured in vitro.Biochim Biophys Acta 915: 254-260

3. Bradford MM (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal Biochem 72: 248-254

4. Grambow HJ, Langenbeck-Schwich B (1983) The relationshipbetween oxidase activity, peroxidase activity, hydrogen perox-ide, and phenolic compounds in the degradation of indole-3-acetic acid in vitro. Planta 157: 131-137

5. Grison R, Pilet P-E (1978) Analyse critique des peroxydases dela racine de mais. Plant Sci Lett 13: 213-218

6. Hinman RL, Lang J (1965) Peroxidase-catalyzed oxidation ofindole-3-acetic acid. Biochemistry 4: 144-158

7. Hoyle MC (1977) High resolution of peroxidase-indoleacetic acidoxidase isoenzymes from horseradish by isoelectric focusing.Plant Physiol 60: 787-793

8. Jacobs M, Rubery PH (1988) Naturally occurring auxin trans-port regulators. Science 241: 346-349

9. Kokkinakis DM, Brooks JL (1979) Hydrogen peroxide-mediatedoxidation of indole-3-acetic acid by tomato peroxidase andmolecular oxygen. Plant Physiol 64: 220-223

10. Kokkinakis DM, Brooks JL (1979) Tomato peroxidase. Purifi-cation, characterization, and catalytic properties. Plant Physiol63: 93-99

11. Magnus V, Iskric S, Kveder S (1971) Indole-3-methanol-ametabolite of indole-3-acetic acid in pea seedlings. Planta 97:116-125

12. Martin HV (1988) Carriers for IAA in maize roots: localisationand possible roles. In M Kutacek, RS Bandurski, J Krekule,eds, Physiology and Biochemistry of Auxins in Plants. Acade-mia Praha, Prague, pp 247-252

13. Martin HV, Beffa R, Pilet P-E (1987) A comparison between3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoicacid. II. Effects on uptake and efflux of IAA in maize roots.Physiol Plant 71: 37-43

14. Martin HV, Pilet P-E (1986) Saturable uptake of indol-3yl-aceticacid by maize roots. Plant Physiol 81: 889-895

15. McCune DC (1960) Multiple peroxidases in corn. Ann NY AcadSci 94: 723-730

16. Mumford FE, Smith DH, Castle JE (1961) An inhibitor ofindoleacetic acid oxidase from pea tips. Plant Physiol 36: 752-756

17. Nakano M, Kobayashi S, Sugioka K (1982) Peroxidase-catalyzedoxidation of indole-3-acetic acid. In M Nozaki, ed, Oxygenasesand Oxygen Metabolism. Academic Press, New York, pp 245-254

18. Ouchterlony 0 (1968) Diffusion in gel. Methods for immunolog-

490 BEFFA ET AL.

www.plantphysiol.orgon April 15, 2020 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.

IAA METABOLITES PRODUCED BY SOLUBLE MAIZE ROOT IAA-OXIDASES

ical analysis. In 0 Ouchterlony, ed, Handbook of Immunodif-fusion and Immunoelectrophoresis. Ann Arbor Science Pub-lishers, Ann Arbor, MI, pp 2-135

19. Pilet P-E (1963) Action de I'acide 2,3,5-triiodobenzoique et duglutathion sur la morphologie et le catabolisme auxinique detissus cultives in vitro. Physiol Plant 16: 299-310

20. Pilet P-E, Lavanchy P (1969) Purification d'extraits peroxyda-siques (Racine de Lens) a activite "auxines-oxydasique." Phys-iol Veg 7: 19-29

21. Pilet P-E, Saugy M (1985) Effect of applied and endogenousindol-3yl-acetic acid on maize root growth. Planta 164: 254-258

22. Pilet P-E, Zryd J-P, Lavanchy P (1967) R6le du pH dansl'analyse de l'activite auxines-oxydasique d'extraits bruts etpurifies. Physiol Veg 5: 327-335

23. Reinecke DM, Bandurski RS (1988) Oxidation of indole-3-aceticacid to oxindole-3-acetic acid by an enzyme preparation fromZea mays. Plant Physiol 86: 868-872

24. Rubery PH (1972) Studies on indoleacetic acid oxidation byliquid medium from crown gall tissue culture cells: the role ofmalic acid and related compounds. Biochim Biophys Acta 261:21-34

25. Rutschmann M, Kuehn L, Dahlmann B, Reinauer H (1982) Highresolution gel chromatography of proteins. Anal Biochem 124:134-138

26. Sabater F, Sanchez-Bravo J, Acosta M (1983) Effects ofenzyme/substrate ratio and of cofactors on the oxidation of indole-3-acetic acid catalyzed by peroxidase. Rev Esp Fisiol 39: 169-

17427. Sembdner G, Gross D, Liebisch H-W, Schneider G (1981) Bio-

synthesis and metabolism of plant hormones. In J MacMillan,ed, Encyclopedia of Plant Physiology (New Series), Vol 9.Springer-Verlag, Berlin, pp 281-444

28. Sequeira L, Mineo L (1966) Partial purification and kinetics ofindoleacetic acid oxidase from tobacco roots. Plant Physiol 41:1200-1208

29. Sundberg B, Sandberg G, Jensen E (1985) Identification andquantification of indole-3-methanol in etiolated seedlings ofscots pine (Pinus sylvestris L.). Plant Physiol 77: 952-955

30. Talwar G, Dendsay JPS, Gupta VK (1985) Kinetic properties ofIAA oxidase from mung bean cotyledons. Phytochemistry 24:673-676

31. Tang YW, Bonner J (1948) The enzymatic inactivation of indole-3-acetic acid. II. The physiology of the enzyme. Am J Bot 35:570-578

32. Tsurumi S, Wada S (1980) Metabolism of indole-3-acetic acidand natural occurrence of dioxindole-3-acetic acid derivativesin Vicia roots. Plant Cell Physiol 21: 1515-1525

33. Vesper MJ, Kuss CL, Maxson JM (1987) The relationshipbetween accumulation of auxin into, and growth of, coleoptilecells. In DJ Cosgrove, DP Knievel, eds, Physiology of CellExpansion during Plant Growth. The American Society ofPlant Physiologists, Rockville, MD, pp 301-303

34. Zmrhal Z, Machackova I (1978) Isolation and characterizationof wheat peroxidase isoenzyme B . Phytochemistry 17: 1517-1520

491

www.plantphysiol.orgon April 15, 2020 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.