Plant Physiol. (1990) 92, 489-4940032-0889/90/92/0489/06/$01 .00/0

Received for publication February 21, 1989and in revised form December 6, 1989

Soluble Peroxidase from Winter Wheat Seedlings withPhenoloxidase-Like Activity1

Maurizio Badiani,* Margherita Gabriella De Biasi, and Marcello FeliciDipartimento di Agrobiologia e Agrochimica, Universita' di Viterbo, Via S.C. De Lellis, 1-01100 Viterbo, Italy

ABSTRACT

Peroxidase isozymes from winter wheat (Triticum aestivum L.cv Orso) seedlings extracts showed phenoloxidase-like activity,becoming visible on polyacrylamide gels also in the absence ofhydrogen peroxide. The results obtained after a characterizationof the two activities, based on their substrate specificity, on theirselective inhibition, and on the possible occurrence of artifacts,suggested the existence of polyfunctional peroxidase isozymes.Different isozymes possessing only phenol oxidase activity werenot found in the same plant material. This appears to be the firstevidence of phenoloxidase-acting isoperoxidases in winterwheat.

POD2 (EC 1.11.1.7) and PO (monophenol monooxygenase,EC 1.14.18.1; laccase, EC 1.10.3.1; diphenol oxidase, EC1.10.3.2) are mostly haeme- and copper-containing glycopro-teins, respectively, which catalyze the oxidation of a largenumber of aromatic structures at the expense of H202 and of02, respectively (22, 24).

In higher plant cells, both POD and PO can occur inmultiple isoforms and are considered to be cytologically andphysiologically different entities (14, 22). PO can exist in alatent state and is thought to be associated to the thylakoidmembrane (22).

It was observed that, upon staining for activity after PAGE,soluble isoperoxidases from winter wheat (Triticum aestivumL. cv Orso) seedlings leaves became visible also in the absenceof H202, thus acting as PO. This unexpected behavior, to-gether with the still open discussion generated by similarobservations in other species (3, 8, 14, 15, 26, 28, 29), led usto examine the nature and the catalytic competence of theseisozymes; these two questions were studied by the action ofsome substances known as effectors of POD or PO and bythe specificity of the two associated activities toward potential

Research work supported by Consiglio Nazionale delle Ricerche,Italy. Special grant Incremento Produttivit6 Risorse Agricole Subpro-ject 1. Paper No. 2094.

2 Abbreviations: POD, peroxidase; PO, phenol oxidase; IAAO,indol-3-yl-acetic acid oxidase; Rubpcase, D-ribulose- 1,5-bisphosphatecarboxylase/oxygenase; CAT, purified catalase from bovine liver;HRP, purified horseradish peroxidase; TYR, purified tyrosinase frommushrooms; L-DOPA, 3,4-dihydroxy-L-phenylalanine; AT, 3-amino-1,2,4-triazole; CTAB, N-cetyl-N,N,N-trimethylammonium bromide;DDTC, diethyl dithiocarbamic acid; SOD, purified superoxide dis-mutase from bovine erythrocytes.

489

hydrogen donors, respectively. Both aspects were studied bymeans of PAGE analysis.

MATERIALS AND METHODS

Reagents

The aromatic compounds used for PAGE activity stainingsand the substances used as effectors ofPOD and PO activitieswere of the highest purity available and were obtained fromAldrich (Steinheim, F.R.G.), BDH (Poole, U.K.), Fluka(Buchs, Switzerland), Merck (Darmstadt, F.R.G.), Serva (Hei-delberg, F.R.G.), and Sigma (St. Louis, MO.); dioxane wasfrom Fluka. Catalase (EC 1.11.1.6) from bovine liver, perox-idase from horseradish, superoxide dismutase (EC 1.15.1.1)from bovine erythrocytes, and tryosinase from mushroomswere from Sigma; BSA was from Merck; Triton X-100 wasfrom Bio-Rad (Richmond, CA.). PAGE chemicals were fromBDH, Bio-Rad, and Sigma.

Plant Material and Tissue Extraction Procedure

The seeds of Triticum aestivum L. cv Orso, purchased froma local market, were soaked for 24 h in running tap water.Swollen seeds were sown on plastic nets suspended on plasticcontainers (about 200 seeds per container). Plants were grownin a controlled-environment chamber (16 h light, 80 W m-2total irradiance, Sylvania GROLUX wide spectrum lamps, at26 ± 1°C/8 h dark, at 18 ± 0.5°C; RH 55%), being suppliedweekly with full strength aerated Hoagland solution [5 ,umoleach Ca(NO3)2 .4 H20 and KNO3, 2 umol MgSO4.7 H20, 1,umol KH2PO4, 0.10 ,umol NaFe-EDTA, 9.1 Mmol MnCl2-4H20, 46.2,mol H3BO3, 0.76 Amol ZnSO4.7 H20, 0.32,molCuSO4.5 H20, and 0.10,umol Na2MoO4 .2 H20 made to 1 Lwith distilled water, pH adjusted to 5.80 with 0.1 M KOH](17).

Shoot samples of 10- to 20-d-old seedlings were collected 2h after the beginning of the photoperiod; 2 g of the shootsamples were ground with a pestle in an ice-cold mortar with8 mL of50 mm sodium phosphate buffer (pH 7.00) containing5 mm 2-mercaptoethanol, 2 mm DTT, 2 mM Na2-EDTA, 0.5mM PMSF, 1% (w/v) BSA, and 1% (w/v) insoluble polyvi-nylpyrrolidone (Polyclar AT). The supernatant obtained afterfiltration by four layers of cheesecloth and centrifugation at39,000g for 30 min was used as an enzymic source. Thisextract was assayed for soluble protein content according tothe method of Bensadoun and Weinstein (2); POD activitywas measured according to the method ofMaehly and Chance(21), using guaiacol as hydrogen donor. In control experi-

Plant Physiol. Vol. 92,1990

ments, Triton X-100 was added to the supernatant (0.05%w/v) and to the corresponding pellet (1% w/v) in order toimprove the extraction of membrane-associated enzyme ac-tivities. After a rapid ultrafiltration by means of UnisepUltracent- 10 cartridges (mol wt cutoff 10 kD, Bio-Rad), intactand Triton-treated fractions were separately submitted tonative PAGE.As indexes of chloroplast disruption, total Chl content and

Rubpcase (EC 4.1.1.39) activity were measured in all frac-tions, following the methods of Moran (23) and of Di Marcoet al. (7), respectively. All procedures were carried out at 4°C.

Native PAGE and Activity Staining Procedures

Anionic slab PAGE was performed under constant current(20 mamp) according to the method of Davis (6); 200 ,ug ofextract soluble protein (corresponding to about 5 POD units)or 150 ,ug of CAT, HRP, or TYR (corresponding to 1650,14.2, or 318.7 units, respectively) were loaded in each slabwell. At the end of the run, each gel slab was submitted toactivity stainings.

Aromatic substrates were previously dissolved, under N2flux, in pure, thoroughly deaerated dioxane and kept in thedark when in solution; each substrate was then added toaqueous Tris, thoroughly deaerated for POD or fully aeratedfor PO stainings: in a final volume of 40 mL, each stainingsolution contained 1.6 mmol substrate, 2 mmol Tris, and 6mL dioxane being the pH adjusted to 7.40. For POD stainings,after 20 min incubation 70 ,umol H202 were added. Theactivity stainings at different pH values, were performed with50 mm sodium acetate (pH 4.50, 5.00, and 5.50) or 50 mMsodium phosphate (pH 6.00, 6.50, and 7.00) buffers.

Selective staining of PO isozymes was attempted followingthe method of Van Loon (29). Wheat IAAO and catalaseisozymes were stained following the procedures of Frenkel(13) and of Clare et al. (5), respectively. Ascorbic acid, AT,CTAB, DDTC, mannitol, oxalic acid and tropolone weredissolved in Tris HCl-dioxane pH 7.40, their final concentra-tions ranging from 4.5 to 10 mM; oxalic acid was also testedat pH 5.50 (25). Gels were dipped into these solutions 20 minbefore adding the staining substrates.CAT (specific activity 1 1,000 units mg protein- ') and SOD

(specific activity 3250 units mg protein-') were dissolved in50 mM Tris-HCl buffer pH 7.40 (1, 20 or 100 ,ug mL-') andleft in contact with the gels for 60 min before staining.The possible uncontrolled production of H202 during PO

staining was checked by dipping control and AT-pretreatedgels in 40 mM L-DOPA and collecting 1.0 mL samples after0, 0.5, 1, 2, 5, 10, 20, and 60 min incubation. These sampleswere then immediately assayed for H202 by the followingmethods: (a) catalytic assay in the presence of CAT (1), (b)formation of colored complexes with titanium (IV) salts (12),and (c) by the procedure of Huang et al. (18) modified asfollows: the samples were added to 1.0 mL of a 0.01% (w/v)solution of FeCl3 plus K3Fe(CN)6, prepared under N2 flux inthe dark immediately before the use. The absorbance of thechromophore resulting from the interaction between H202and the ferric salts was measured at 745.2 nm after 24 hincubation in stoppered tubes in the dark and compared witha previously determined calibration curve. This last method

was the most sensitive, 1 AM H202 being the lowest detectableconcentration.Each activity staining and any other procedure on gels was

replicated twice.

RESULTS AND DISCUSSION

The results reported here refer mainly to soluble and/or tolightly membrane bound POD/PO isozymes.

Intact and Triton X- 100-treated fractions of the crudesupernatants exhibited similar total Chl content (about 35 ,ugmg protein-') and Rubpcase activity (about 4 units mg pro-tein-'). In the corresponding pellet, the respective values wereabout 110 ,g mg protein-' and about 0.8 units mg protein-'.A complete POD isozymic distribution after PAGE is

shown in Figure 1. The pattern did not appear when wheatextracts were heated in boiling water for 2 min, submitted toPAGE, and then stained or when, after PAGE, gels wereboiled for 10 min before being stained.

All the compounds followed by the notation '+' in Table Icaused, in 30 to 60 min, the staining of at least one of theisozymes shown in Figure 1, multiple plus indicating anincreased pattern intensity. A much slower visualization (8-10 h) was observed with L-tyrosine.Table I shows that the range ofaromatic structures effective

a ---b -- VLMC-

d-e- LM

- II

HM

3

Rm0.00

-0.25

-0.50

0.75

-1.00

Figure 1. Anionic disc PAGE pattern (separating gel) of T. aestivumleaves isoperoxidases stained with guaiacol and hydrogen peroxide.VLM, very low mobility; LM, low mobility; IM, intermediate mobility;HM, high mobility. Rm, relative electrophoretic mobility.

490 BADIANI ET AL.

-le

PHENOLOXIDASE-ACTING ISOPEROXIDASES IN WINTER WHEAT

Table I. Effectiveness of Different Substrates for the Staining of Triticum aestivum Zymograms in the Presence (POD) or in the Absence (PO) ofH202

Letters indicating isoforms stained are as in Figure 1. IUPAC names of the compounds are reported in the footnote.

Substrate POD PO Substrate POD PO

No -OH/-NH2 group One -OH and one -OR group1. cinnamic acid - - 27. guaiacol +++ (All) + (a-c)

One -OH/-NH2 group 28. 4-methoxyphenol + (All)2. phenol + (a-c; j-p) - 29. 4-methoxy-1 -naphtol ++ (All)3. aniline ++ (a-f) + 30. guaethol ++ (All) + (a-c)4. 4-hydroxybenzoic acid + (a) _ 31. 4-propoxyphenol + (All)5. 3-hydroxyphenylacetic acid + (a) _ 32. vanillic acid + (a-f) -6. p-coumaric acid + (a-f) - 33. ferulic acid ++ (All) + (a-c)7. L-tyrosine - + (a) 34. methyl vanillate ++ (a-h)8. umbelliferone + (a-c) - Two -OR groups

Two -OH/-NH2 groups 35. veratrole + (a)9. cathecol ++ (All) + 36. veratryl alcohol

10. resorcinol ++ (All) + One -OH and two -OR groups11. hydroquinone ++ (All) + 37. 2,6-dimethoxyphenol ++ (All) + (-p)12. p-naphthohydroquinone ++ (All) + 38. syringaldehyde + (a)13. o-phenylenediamine +++ (All) ++ 39. syringic acid ++ (All) +14. esculetin + (a-c) + (g-h) 40. sinapic acid ++ (All) +15. 3,3'-diaminobenzidine ++ (a-f; j-p) + 41. syringaldazine + (a) +16. protocatechuic acid ++ (All) + Different compounds17. 2,3-dihydroxybenzoic acid ++ (All) + (d-f) 42. tropolone +(a-f)18. gentisic acid + (a-f) + 43. 4-DMACA19. chlorogenic acid ++ (All) + (g-h) 44. IAA:20. 3,4-dihydroxyphenylacetic acid ++ (All) + a) direct staining21. caffeic acid ++ (All) + b) Frenkel's method ++ (All)22. hydrocaffeic acid ++ (All) + 45. 2,4-DCP ++ (All) + (a-f)23. L-DOPA ++ (All) ++ 46. ABTS + (a-f) +

Three -OH groups24. pyrogallol ++ (All) +25. phloroglucinol + (All) ± (traces)26. gallic acid ++ (All) +

1. 3-phenylacrylic acid; 6. 4-hydroxycinnamic acid; 7. 2-amino-3(4-hydroxyphenyl)propionic acid; 8. 7-hydroxycoumarin; 9. 1,2-dihydroxy-benzene; 10. 1,3-dihydroxybenzene; 11. 1,4-dihydroxybenzene; 12. 1,4-dihydroxynaphthalene; 13. 1,2-diaminobenzene; 14. 6,7-dihydroxy-coumarin; 15. 3,3',4,4'-tetraaminobiphenyl; 16. 3,4-dihydroxybenzoic acid; 18. 2,5-dihydroxybenzoic acid; 19. 3-0(3,4-dihydroxycinnamoyl-D-quinic acid; 21. 3,4-dihydroxycinnamic acid; 22. 3,4-dihydroxyphenylpropionic acid; 23. 3-3,4-dihydroxyphenyl-L-alanine; 24. 1,2,3-trihy-droxybenzene; 25. 1,3,5-trihydroxybenzene; 26. 3,4,5-trihydroxybenzoic acid; 27. 2-methoxyphenol; 30. 2-ethoxyphenol; 32. 4-hydroxy-3-methoxybenzoic acid; 33. 4-hydroxy-3-methoxycinnamic acid; 34. methyl-4-hydroxy-3-methoxybenzoate; 35. 1 ,2-dimethoxybenzene; 36. 3,4-dimethoxybenzyl alcohol; 38. 3,5-dimethoxy-4-hydroxybenzaldehyde; 39. 3,5-dimethoxy-4-hydroxybenzoic acid; 40. 3,5-dimethoxy-4-hydrox-ycinnamic acid; 41. 4-hydroxy-3,5-dimethoxybenzaldehyde azine; 42. 2-hydroxy-2,4,6-cycloheptatrien-1-one; 43. 4-dimethylamino-cinnamal-dehyde; 44. 3-indolylacetic acid; 45. 2,4-dichlorophenol; 46. 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid.

in producing PAGE zymograms was wider in the presencethan in the absence of H202. PO-stained isoforms showed thesame mobility of the corresponding ones stained for PODactivity but the presence of H202 caused an intensification ofthe pattern. This last effect was not observed by adding H202to PO-stained gels previously heated at 100°C for 10 min.

Intact and Triton-treated extracts did not differ in theirPOD/PO zymograms stained with guaiacol or L-DOPA, re-spectively. With the same hydrogen donors, ultrafiltration ofthe crude extracts did not modify the POD/PO zymogramsobtained.Some relationships between substrate structures and the

staining of PO-acting isoperoxidases can be inferred fromTable I:

(a) The presence of at least one phenolic group appeared tobe essential for the visualization of PO-acting isoforms (Nos.

1 and 43). The results obtained with veratrole (No. 35) makethis restriction not valid in the presence of H202; in this case,VLM a-POD isoform should have necessarily acted as anoxygenase. In theory, the presence of etherified groups couldhave favored this hydroxylation while the presence of addi-tional groups (No. 36) could have prevented it to occur.

(b) In the presence of only one phenolic group, PO-actingisoforms were undetectable (Nos. 2, 4-6, and 42), an excep-tion being observed with L-tyrosine (No. 7), When an aminogroup replaced the phenolic one, some isozymes becamevisible also in the absence of H202 (No. 3).

(c) PO-acting isoforms became visible when two free phe-nolic groups were present; their relative position on the aro-matic ring did not influence the number of isoforms stained(Nos. 9-12) as well as a carboxyl group, linked directly (No.16), or through saturated or unsaturated side chains (Nos.

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Plant Physiol. Vol. 92,1990

20-23) to the aromatic ring. However, when one of thephenolic group was in ortho position to the carboxyl one(Nos. 17 and 18) or when the carboxyl group was intramolec-ularly (No. 14) or intermolecularly (No. 19) esterified, incom-plete PO patterns appeared.When the second phenolic group was etherified, PO-acting

isoforms were detectable even if their substrate affinity ap-peared to be reduced when compared with the parent diphen-ols and only VLM isoforms were visualized (Nos. 27, 30, and33). When the etherified (Nos. 28, 29, and 31) or esterified(No. 8) phenolic group was in para position to the free one,no evidence ofPO staining was obtained. The size of the alkylgroups which etherified the phenolic one appeared to be notrelevant (No. 30). If a side chain was present, PO-actingisoforms remained visible (No. 33). On the other hand, acarboxyl group linked directly to the aromatic structure pre-vented PO isozymes to appear (Nos. 32 and 34).

(d) When three phenolic groups were present, PO-actingisoforms became stained, the relative position of phenolicgroups having a strong effect on the number of isoformsstained (cf Nos. 24 and 26 with No. 25). The methylation oftwo of the three phenolic groups reduced the number of PO-acting isoforms stained (Nos. 37 and 41) while no reductionwas observed when a carboxyl group was linked directly (Nos.26 and 39) or through a short side chain (No. 40) to thearomatic ring. The presence of an aldehydic group preventedPO pattern to appear (No. 38).

(e) The substitution of hydroxyl groups with amino ones(Nos. 3, 13, and 15) improved the effectiveness ofthe aromaticsubstrates for the visualization of both POD and PO-likezymograms (cf No. 3 with No. 2 and No. 13 with No. 9).

(f) By following the staining procedure described by Frenkel(13), IAAO activity pattern was found to be identical to thezymogram of Figure 1 (No. 44 in Table I). However, 2,4-dichlorophenol, which is employed in the coupled oxidationleading to IAAO visualization, was itself effective in stainingall POD isozymes and slow-migrating PO-acting isoforms(No. 45); this evidence casts some doubt on the specificity ofthe IAAO-staining procedure.

(g) Both enzymic functions possessed the ability of abstract-ing an hydrogen atom from ABTS molecule (No. 46), prob-ably leading to the formation of a resonance-stabilized chro-mogenic radical cation.

(h) The results obtained with L-tyrosine (No. 7) broke tworules: it was the only hydrogen donor which caused theappearance of VLM-a PO-acting isoform but not of thecorresponding POD one. Also, it was the only monophenolicstructure effective as staining substrate in the absence ofH202.It is known that tyrosinase, acting as an oxygenase, hydrox-ylates L-tyrosine to 3,4-L-DOPA which can be oxidized toDOPA-quinone and finally incorporated into a chromogenicpolymer. On the other hand, peroxidase-peroxide system actson L-tyrosine by forming an o-o'-biphenyl-linked dimer ('di-tyrosine') (27), which could not be a good candidate to formchromogenic polymers.To explain the anomalies observed with L-tyrosine, it is

proposed that in the absence of H202, VLM-a isoform actedas a tyrosinase by transforming L-tyrosine into L-DOPA, beingthis last oxidized either by VLM-a itself, with a diphenoloxi-

dase mode of action, and/or nonenzymically. The final resultcould have been a slow visualization of VLM-a, as it actuallywas. On the other hand, in the presence of H202 VLM-acould have acted as a peroxidase, an achromogenic and/orwater soluble oxidation product being formed.To verify the nature of the supposed bifunctional POD-

isoforms, the possible occurrence of artifactual PO stainingand the discriminating action of some effectors ofPO activitywere evaluated (Table II and III).

Artifactual PO staining could have been developed in thepresence of peroxide traces in the incubation media (29) and/or if H202 had been produced by the spontaneous autoxida-tion ofhydrogen donors in the presence ofwater (27). Besides,PODs can form H202 providing that suitable electron donors,such as IAA, NAD(P)H or sulfite, and Mn>2 ions were present.This reaction proved to be inhibited by free superoxide radicalscavengers, such as dihydroxyphenols and SOD, and stimu-lated by monophenols (9, 16). Once formed, this H202 couldhave been utilized by POD itself, producing false PO activitystainings.

It should be considered, however, that: (a) by employingthree different assay procedures, no evidence ofH202 produc-tion was obtained during the staining of the gels for POactivity. This same result was obtained also when endogenouscatalase was inhibited by the presence of AT (1); (b) a largeexcess of added CAT did not prevent the staining of PO-acting POD isozymes (Table II), though reducing the speedand intensity of their visualization. This effect, reversed bythe presence of AT, was explained in terms of competitionfor the hydrogen donor since also CAT isozymes were foundable to oxidize L-DOPA in the absence of H202 (data notshown). These PO-acting CAT isozymes were the same whichstained intensely in the presence ofguaiacol and H202; (c) thepresence of electron donors and metal cofactors in the gelswas thought to be unrealistic after PAGE. Besides, bothdihydroxyphenols (Table I) and SOD (Table II) failed to affectPO-staining ofPOD isozymes.

Thus, the reported results suggest that H202 was not in-volved in the visualization of PO-acting isoperoxidases.The autoxidation of hydrogen donors could also have lead

to the production of partially reduced 02 species, such as 02and/or OH, promoting the formation of chromogenic poly-mers via radical chain reactions. However, dihydroxyphenols(Table I) are considered powerful scavengers of activatedoxygen species (9) and both mannitol, a proper OH scavenger(4), and SOD did not affect the activity stainings (Table II).Hence, it can be suggested that activated oxygen specieswere not involved in the visualization of PO-acting PODisoforms. This confirms the conclusions of Yamazaki andYamazaki (30) and ofKahn et al. (19) concerning the reactionmechanisms of POD-IAAO system and of cathecol oxidase,respectively.As reported in Table III, changing the staining pH or using

a specific staining procedure (29) did not evidentiate anygenuine PO isoform.

Ascorbic acid and DDTC, typical copper chelators (22), didnot prevent the staining of the zymograms in the absence ofH202 (Table III). This result confirms the observations ofGrison and Pilet (15) on the PO-acting maize root PODs. The

492 BADIANI ET AL.

PHENOLOXIDASE-ACTING ISOPEROXIDASES IN WINTER WHEAT

Table II. Analysis of Artifactual Staining of PO-Acting POD from T. aestivum Leaves due toUncontrolled Production of H202 and/or Partially Reduced 02 Species

Zymograms stained with guaiacol plus H202 (+H202) or with L-DOPA plus 02 (-H202).

Substance Added to the Staining Effects on ZymogramsMediaaMediaa ~~~+H202 -H202

AT Slight hastening (15-30 min) NoneCAT NDb Slight to strong delaying and

fading increasing with con-centration

CAT + AT ND Slight delaying and fadingMannitol None NoneSOD None Very slight delaying and fading

a Concentrations used: AT, 10 mM; CAT, 0.024, 0.48, or 2.4 ttM; CAT + AT, 24 nm + 20 mm or 480AM + 400 mM; mannitol, 5 mm; SOD, 3 or 30 ,M. b Not determined.

Table Ill. Selective Visualization of PO and Identification of PO-Acting POD from T. aestivum LeavesZymograms stained as in Table II.

Effects on ZymogramsTreatmenta

+H202 -H202

Incubation at pH 4.5-7.4 None NoneSelective PO staining NDb NoneAscorbic acid Slight delaying and fading Strong delaying and fadingCTAB Slight hastening Slight hasteningDDTC Slight delaying and fading Strong delaying and fadingOxalic acid None NoneTropolone Slight fading Slight fading with L-DOPA

None with L-tyrosinea Concentrations used: Ascorbic acid, 6.0 mM; CTAB, 5.0 mM; DDTC, 4.5 mm; oxalic acid, 5.0 mM;

tropolone, 5.0 mM. b Not determined.

inhibitory effect of ascorbic acid and DDTC on POD andPO-like zymograms could have been due to an aspecificscavenging of the radical quinonoid species arising from theenzymic oxidation of the substrates (29). Oxalic acid, whichwas found to inhibit PO from spinach (25), did not preventthe visualization of the PO-acting isoforms, both at neutraland at acidic pH values. CTAB, thought to be a laccaseinhibitor (22), did not prevent the oxidation of selectivelaccase substrates, such as syringaldazine or 2,6-dimethoxy-phenol, in the absence of H202.Another line of evidence supporting the existence of proper

POD isozymes with PO-ability was obtained with tropolone(Table III). Kahn (20) showed that tropolone is a powerfulinhibitor ofTYR and is a good substrate for HRP. Thus, theuse of this molecule to differentiate the two activities in plantextracts was suggested. In the present experiments, afterPAGE TYR revealed at least 10 isoforms in the presence ofguaiacol or L-DOPA. The addition of H202 did not affectboth the intensity and the visualization speed of the pattern.With L-tyrosine as substrate, only intermediate-mobility iso-forms appeared; their visualization was completely preventedby a preincubation of the gels with 5 mM tropolone. However,tropolone did not inhibit L-tyrosine oxidation brought aboutby VLM-a isoform (Table III). This could further confirm itsnature of proper and bifunctional POD isozyme.

PO-acting isozymes were also present in pure commercial

POD preparations (HRP): with L-DOPA as substrate, at leastsix slow migrating isoforms stained both in the presence and,less intensely, in the absence of H202.

CONCLUSIONS

The data reported here appear to be the first evidence ofPOD isozymes which can all behave as PO (with tyrosinase,laccase, and diphenoloxidase activities) and as IAAO in T.aestivum leaves.The range of aromatic structures which acted as substrates

of the PO-like function was comparable to that of proper andfully characterized PO from fungi (1 1) and appeared to bewider than that of PO-acting isoperoxidases from maize roots(15).The physiological implications, if any, of POD polyfunc-

tionality remain unknown. However, this property could beof use under some conditions; for example, during ligninbiosynthesis, the peroxidase- (16) or amino oxidase-mediated(10) H202 formation could become locally and/or transientlylimited by a reduced availability of electron donors and/orcofactors. In this situation, a hypothetical PO-acting cell-wallPOD could help, as being capable of utilizing also 02 tocatalyze the free-radical mediated condensation of cinnamylderivatives.The apparent lack ofgenuine PO isozymes in winter wheat

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leaves is remarkable. However, only latent and thus unde-tectable PO isoforms could be present in young and healthytissues, grown under favorable and controlled conditions.

ACKNOWLEDGMENTS

The authors are greatly indebted to Professor J. M. Bollag and hiscolleagues at Pennsylvania State University for reviewing the manu-

script. The continuous support and the helpful suggestions of Profes-sor G. Giovannozzi Sermanni and Professor V. Buonocore are grate-fully acknowledged, as well as Dr. C. Perani and Miss A. R. Paolaccifor drawings.

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