Fifth European Fusarium Seminar

Szeged, Hungary, 1997

POLYPHENOL OXIDASE IN FUSARIUM OXYSPORUM F. SP. DIANTHI: PARTIAL CHARACTERIZATION AND ACTIVITY
P. CURIR, A. MERCURI, F. D'AQUILA, C. PASINI, and M. DOLCI* Istituto Sperimentale per la Floricoltura, Corso Inglesi 508, 18038 Sanremo *Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali - Universit di TorinoItaly Abstract A polyphenol oxidase has been extracted and partially purified from Fusarium oxysporum f. sp. dianthi cultivated in vitro. The enzyme biosynthesis was enhanced by the addition to the medium growth of 3-hydroxyacetophenone. Four different enzyme forms were separated through electrophoresis, and their apparent molecular mass has been calculated. The enzyme proved to possess a catechol oxidase activity, but not the cresolase ODe; its oxidative activity has been tested on different substrates. Introduction Polyphenol oxidase (PPO) has been particularly investigated in the higher plants (Flurkey, 1986) but only few data exist about this enzyme in the fungi where, moreover, its precise pathogenic role has not been determined (Mayer and Harel, 1979). This enzyme has been extensively studied in the Neurospora fungus (Lerch, 1983) and, although Fusarium oxysporum proved able to produce PPO (Mace and Wilson, 1964; Maraite, 1973), no studies bave been carried out about its presence in F. oxysporum f. sp. dianthi, likely because of the purification difficulty (Mayer and Harel, 1979). The present research had thus the aim of investigating the PPO in the latter F. oxysporum f. sp. dianthi, through a partial purification which allowed the characterization of some intrinsic properties of the enzyme and an evaluation of its oxidative activity towards different phenolic substrates. Material and methods Protein extraction - F. oxysporum f.sp. dianthi pathotype 2 was grown on potato dextrose agar (PDA) medium, alone (control) or with the addition of the phytoanticipin 3-hydroxyacetophenone (3-HAP) 10 mM L-l (Curir et al., 1996), in Petri dishes (10 cm diameter). The inoculum for each plate consisted of 1 cm diameter mycelium disk; 200 dishes were prepared both for the control and for the treatment. The fungus was allowed to grow for 15 days on PDA alone and on the treated medium. Every further step of purification has been carried out at 4C. The developed mycelium and the colonized gelled medium were respectively collected and homogenized in cold acetone with a Turrax blender (10 g material/50 mL acetone). The two obtained homogenates were then filtered on cheesecloth, washed three times with acetone, centrifuged at 5,000 rpm for half an hour and the respective sediment allowed to completely dry. To the dried material, sodium phosphate buffer 0.1 M, pH 7.0, (l g material/40 mL buffer) and 5 g L-l polyvinylpirrolidone were added. The obtained suspensions were stirred for three hours always at 4 cC, filtered through cheesecloth, centrifuged as above and then the respective sediments were discarded. 600 g L-l ammonium sulphate were added to both the clear solutions and the precipitated proteins were collected by centrifugation at 6,000 rpm. The precipitates obtained both from the contrai and from the treated

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mycelium were then redisso1ved with the buffer, desaIted through dialysis and concentrated to 20 mL with a NucIepore apparatus having a 5 kDa cut off membrane. Enzyme partial purification - The protein extract from the treated mycelium was first chromatographed on a Deae-Cellulose column (50 x 0.8 cm), packed with the already mentioned buffer and eluted with a linear gradient from pure buffer to 15% sodium chloride in buffer. The enzyme activity of the eluate was spectrophotometrically monitored with 4-methyIcatechol (4-MC) 0.5% in buffer, reading the Abs changes at 400 nm, and the active fractions were pooled, dialyzed and concentrated as above. The pooled enzymatic fraction was rechromatographed onto a Sephacry1 S-300 column (50 x l cm), packed and eluted with the buffer. The enzymatically active fractions were pooled and concentrated as already mentioned. These solutions represented the partiaIly purified enzyme. Enzyme assay and evaluation of its activity towards different phenolic substrates - The standard oxidative activity of the enzyme was spectrophotometrically measured with 4-MC dissolved in the buffer in different concentrations, following the formation of 4-methyl-o-benzoquinone at = 400 nm (E = 1350 M-l cm-l), and considering as l Unit of enzyme activity the amount of enzyme able to produce 1mo1e of 4-methyl-o-benzoquinone per minute (Sanchez-Ferrer et al., 1988). When different phenols were used as substrates, they were tested in 5 mM concentration in the buffer; the rate of oxidation was caIculated using the known absorbance maximum and molar extinction coefficient appropriate for each substrate. In order to compare the enzyme activity towards 4-MC with that towards other substrates, the disappearance of l mole of each compound was considered as l Unit of enzyme activity towards each specific compound. Protein quantitation - Protein concentration was determined with the method of Bradford (1976), using bovine serum albumin as a reference. Isoelectrofocusing (IEF) analysis - IEF of the partially purified enzyme was performed with horizontal electrophoresis on 1% agarose slabs (l0 x l0 cm, 500 m thickness) containing Biorad ampholytes to generate a pH gradient from 3.0 to 9.0. Agarose gels contained 10% sorbitol. Electrophoretica1 runs were performed using 0.1 N acetic acid as the anodic solution, while the cathodic solution was 0.1 N sodium hydroxide. Refrigeration plate temperature was 5C. The samples were loaded onto the gel slabs as droplets (10 L) poured onto Whatman paper squares (0.8 x 0.8 cm), which were put in the middle part of the slabs. Run parameters were: 400 V current, l0 mA intensity, and 8 W power. Each run had a 1.5 hr duration. The fixative solution was composed of 30% methanol, 5% trichloroacetic acid, 3.5% sulphosalicylic acid, and 61.5% water. The slab staining for the proteinograms was performed using a solution of 0.2% Coomassie blue, 14% actic acid, 30% ethanol, and 55.8% water; the Coomassie blue was replaced by 0.5% 4-MC in the buffer when the enzymatic activity needed to be located (zymograms). IEF markers used were: lentil lectin (pI 8.20-8.60), human haemoglobin c (pI 7.50), human haemoglobin a (pI 7.10), equine myoglobin (pI 7.0), human carbonic anhydrase (pI 6.50), bovine carbonic anhydrase (pI 6.0), -lactoglobulin b (pI 5.10) and phycocyanin (pI 4.65). Polyacrylamide gel electrophoresis (PAGE) - PAGE analyses were carried out on dry pre-casted Clean Gel (Pharmacia) polyacry1amide slabs, rehydrated according to the manufacturer's instructions. Twenty mL of sample solution were applied, with the following run parameters: 300 V current, 18 mA intensity and l0 W power. The molecular mass markers were: bovine erythrocyte carbonic anhydrase (29 kDa), chicken egg albumin (45 kDa), bovine albumin (66 kDa), phosphorylase b (97.4 leDa), Escherichia coli -galactosidase (116 kDa), rabbit myosin (205 kDa). Slabs were stained with Coomassie blue.

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Fifth European Fusarium Seminar Results and discussion

Szeged, Hungary, 1997

The investigated fungal enzyme proved to be very difficult to purify, due to the interactions with contaminant phenols, which likely modify or inactivate this protein through the forrnation of hydrogen, ionic, and covalent bonds (Loomis, 1974). These difficulties, typical for plant PPOs (Kimberly and Montgomery, 1985), aIlowed us to obtain only a parti al purification of the enzyme which gave a reduced recovery of the activity (Table 1). 3-hydroxyacetophenone acted as a specific inducer nf the enzyme, although it did not behave as a substrate (Table 2). Even the other assayed monophenols were not oxidizable by the enzyme, while the other three tested o-diphenols were good substrates (Table 2). This seems to indicate that the enzyme is characterized by a catechol oxidase activity but is deprived of a cresolase activity, which is typical for the polyphenol oxidase of the plants (FIurkey, 1985). The purified enzymatic fraction showed folle electrophoretical bands: a main cathodic band, with a pI around 7.5, a neutral band at pI 7.0 and two anodic bands between 6.0 and 6.5 (Figure 1). AlI these folle forrns proved to possess a polyphenol oxidase activity when the electrophoretical slabs were tested for the enzymatic activity. PAGE analyses of the protein extracts from the mycelium developed on PDA alone or in the presence of 3-HAP, showed that the PPO biosynthesis is greatIy enhanced by this phenol: the zymograms revealed enzymatic bands detectable only in traces in the control extracts, while in the treated samples deeply stained bands were appreciable (Figure 2). Four enzyme forrns were detectable, with an apparent molecular mass of 45, 50, 66, and 90 kDa respectively. The 45 kDa forrn should correspond to the 42 kDa functional unir usuaIly found in ali the studied fungal PPOs (Mayer and lIarel, 1979); the other forrns may come from enzyme association-dissociation which could originate a tetramer-monomer interconversion, as frequentIy reported in literature for this kind of enzyme (Mayer and lIarel, 1979). The F. oxysporum f. sp. dianthi PPO seems to be essentiaIly an indcible enzyme, the synthesis of which can be possibly stimulated by the phenols present in the attacked plant tissues. Due to its catechol oxidase activity, this enzyme may potentiaIly participate in vascular browning of the diseased tissues which takes piace upon the activity of plant PPO. This latter characteristic could be analogous to that reported for F. oxysporum f. sp. cubense PPO (Mace and Wilson, 1964). The detection of enzyme different forms with a dose apparent molecular mass does not allow to argue if monomers afe associated to originate a further quatemary structure or if they represent isoenzymatic aspects of the same physiological unit.. The multiplicity of enzyme electrophoretical bands is comparable to that observed in grape catechol oxidase by Lemer et al. (1972), and as for the case described by the authors it is also possible that the bere considered enzyme undergoes changes in aggregation amI/or in conforrnation, making the investigations more and more difficult. Further studies wiIl thus bave the aim of improving the purification protocol to allow a more complete study of this enzyme. Table I. Partial purification of Fusarium oxysporum f. sp. dianthi PPO. Purification step Crude extract Deae-cellulose Sephacryl S-300 Volume (rnL) 1,500 50 10 Total protein (mgfrnL) 2.1 3.3 7.0 Specific activity (Units/mg protein) 1.7 15.0 32.0 Purification fold 1 9 19

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Figure 1. IEF of the partially purified PPO enzyme. Right lane indicates the pI markers. Arrow indicates the neutral pH area.

Figure 2. PAGE of the partially purified PPO. a: enzyme fractions; b: molecular mass markers

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Fifth European Fusarium Seminar

Szeged, Hungary, 1997

Table 2. Partially purified enzyme activity determined in buffer, at a 5 mM substrate concentration; the reaction mixture included 1 g/mL PPO in a cuvette of 2.5 mL. Activity expressed as percent of activity compared with 4-methylcatechol assumed as 100%.

Assayed substrate 3- h ydroxy acetophenone monophenols p-coumaric acid vanillic acid 4-methylcatechol o-diphenols caffeic acid chlorogenic acid protocatechuic acid

Wavelenght (nm) 249 287 260 400 325 325 293

Relative activity (%) 0 0 0 100 60 70 45

Literature Bradford, M. M. 1976. A rapid and sensitive method far the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72:248-254. Curir, P., Marchesini, A., Danieli, B., Mariani, F. 1996. 3-Hydroxyacetophenone in camations is a phytoanticipin active against Fusarium oxysporum f. sp. dianthi. Phytochemistry 41:447-450. Flurkey, W. H. 1985. In vitro biosynthesis of Vicia faba polyphenol oxidase. Plant Physiol. 79:564-567. Flurkey, W. H. 1986. Polyphenol oxidase in higher plants. Plant Physiol. 81 :614-618. Kimberly, W. W., Montgomery, M. W. 1985. Purification of d'Anjou Pear (Pyrus communis L) polyphenol oxidase. Plant Physiol. 78:256-262. Lerch, K. 1983. Neurospora tyrosinase: structural, spectroscopic and catalytic properties. Mol. Cell Biochem. 52:125-138. Lerner, H. R., Mayer, A. M., Harel, E. 1972. Evidence far conformational changes in grape catechol oxidase. Phytochemistry 11:2415-2421. Loomis, W. D. 1974. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol. 31:528-544. Mace, M. E., Wilson, E. M. 1964. Phenol oxidases and their relation to vascular browning in Fusarium-invaded banana roots. Phytopathol. 54:840-842. Maraite, H. 1973. Changes in polyphenol oxidases and peroxidases in muskmelon (Cucumis melo L) infected by Fusarium oxysporum f. sp. melonis. Physiol. Plant Pathol. 3:29-49. Mayer,A., Harel E. 1979. Polyphenol oxidases in plants. Phytochemistry 18:193-215. Sanchez-Ferrer, A., Bru, R., Cabanes, J., Garcia-Carmona, F. 1988. Characterization of catecholase and cresolase activities ofMonastreli grape polyphenol oxidase. Phytochemistry 27:319-321.

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