Carbajoetal2002

Tannic acid induces transcription of laccase genecglcc1 in the white-rot fungus Coriolopsis gallica

José M. Carbajo, Howard Junca, María C. Terrón, Tania González, Susana Yagüe,Ernesto Zapico, and Aldo E. González

Abstract: Laccase, a phenoloxidase enzyme secreted by white-rot fungi, has a significant role in the degradation oflignin and environmental pollutants. Coriolopsis gallica is a ligninolytic basidiomycete that produces high levels of thisextracellular enzyme. A laccase gene cglcc1 from this fungus has been cloned and sequenced. The capacity of C.gallica to efficiently degrade polyphenols has been successfully applied in our laboratory to the biotreatment anddecolorization of several industrial wastewaters. This study focused on the effect of tannic acid, a natural compoundwidely distributed in plants, on the production of laccase activity by C. gallica. Our results showed an evident increaseof extracellular laccase levels when C. gallica was grown in the presence of tannic acid. Concentrations of 50 and100 µM of this compound increased laccase activity when compared with control samples grown without tannic acid.In addition, we found an increase in laccase transcript levels in C. gallica grown in culture media supplemented withtannic acid. The role of tannic acid was shown to be an inductor of laccase activity in this fungus, due to the enhance-ment of expression of the laccase gene at the transcriptional level.

Key words: laccase, tannic acid, Coriolopsis gallica, induction, gene transcription.

Carbajo et al.Résumé : La laccase est une enzyme phénoloxydase secrétée par les champignons responsables de la pourritureblanche et qui joue un rôle significatif dans la dégradation de la lignine et de divers polluants environnementaux. LeCoriolopsis gallica est un basidiomycète qui produit des quantités élevées de cette enzyme extracellulaire. Un gènecglcc1 de la laccase de ce champignon a été cloné et séquencé. La capacité de C. gallica à dégrader efficacement lespolyphénols a été appliquée avec succès dans notre laboratoire pour le biotraitement et la décoloration de quelqueseaux usées industrielles. La présente étude a vérifié l’effet de l’acide tannique, un produit naturel largement répanduchez les plantes, sur la production de l’activité laccase par C. gallica. Les résultats obtenus ont démontré une netteaugmentation des niveaux de laccase extracellulaire lorsque C. gallica était cultivé en présence d’acide tannique. Desconcentrations de 50 et 100 µM de ce produit ont fortement augmenté l’activité laccase comparativement à des échan-tillons de contrôle cultivés en absence d’acide tannique. Nous avons de plus constaté une augmentation des niveaux detranscription de la laccase chez C. gallica cultivé dans des milieux enrichis d’acide tannique. Nous démontrons ainsi lerôle de l’acide tannique comme inducteur de la laccase chez ce champignon à cause d’une augmentation del’expression du gène et de la transcription de la laccase.

Mots clés : laccase, acide tannique, Coriolopsis gallica, induction, transcription d’un gène.

[Traduit par la Rédaction]1047

Introduction

White-rot basidiomycetous fungi are gaining interest be-cause of their capability to degrade a wide variety of naturaland synthetic materials and environmentally persistentorganopollutants, such as chlorophenols, polycyclic aromatichydrocarbons, polychlorinated biphenyls, and industrial dyes

(Pointing 2001). A highly nonspecific ligninolytic enzymaticsystem secreted by white-rot fungi is known to be involved inthis biodegradation (Thurston 1994; Leonowicz et al. 1999).Research on this topic has been undertaken recently in manylaboratories because of its great potential in several biotech-nological applications, such as bioremediation (Roy-Arcandand Archibald 1991; Pointing 2001), animal feed improve-

Can. J. Microbiol. 48: 1041–1047 (2002) DOI: 10.1139/W02-107 © 2002 NRC Canada

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Received 8 July 2002. Revision received 20 November 2002. Accepted 28 November 2002. Published on the NRC Research PressWeb site at http://cjm.nrc.ca on 7 January 2003.

J.M. Carbajo, H. Junca,1 M.C. Terrón, T. González,2 S. Yagüe, E. Zapico,3 and A.E. González.4 Departamento deMicrobiología Molecular, Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC),Velázquez 144, E-28006, Madrid, Spain.

1Present address: Department of Environmental Microbiology, Gesellschaft für Biotechnologische Forchung (GBF)-NationalResearch Centre for Biotechnology, D-38124 Braunschweig, Germany.

2Present address: Instituto Cubano de Derivados de la Caña de Azúcar, Havana, C.P. 11000 Cuba.3Present address: Biotechnology Department, University of Hamburg, Hamburg, D-21073 Germany.4Corresponding author (e-mail: [email protected]).

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ment (Akin et al. 1993), pulp and paper production (Messnerand Srebotnik 1994), and wastewater treatment (Terrón et al.1993; Garg and Modi 1999).

Studies on lignin-degrading enzymes have been mainly fo-cused on peroxidases (lignin and manganese peroxidases) andlaccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2).Although biochemical, genetic, and regulatory aspects ofperoxidases have been relatively well studied (Cullen 1997),information on laccases is still scarce. Nevertheless, the im-portant role of laccases in degradation of lignin and otherphenolic molecules has been confirmed (Eggert et al. 1997).

To date, there are few reports focused on the regulatorymechanisms involved in laccase production (Collins andDobson 1997; Mansur et al. 1998; Palmieri et al. 2000).Still, these studies are of interest given that large quantitiesof the enzyme will be required for further practical applica-tions in bioremediation and biotechnology (Cullen 1997).There are several studies demonstrating increased laccaseactivity levels in basidiomycetes caused by natural andsynthetic aromatic substances (Pickard and Westlake 1970;Arora and Sandhu 1984). In contrast, there are few reportson the effect of phenolics and other substances on theenhancement of laccase gene transcription (Linden et al.1991; Collins and Dobson 1997; Mansur et al. 1998).Some of these inducers are synthetic molecules, such as 1-hydroxybenzotriazole, cycloheximide, and 2,5-xylidine,which show a significant degree of toxicity to animal andhuman health. Given the toxicity associated with these sub-stances, the search for less harmful compounds that are ableto increase laccase levels is of environmental importance.

The ligninolytic basidiomycete Coriolopsis gallica hasbeen selected in our laboratory, based on the results of aprevious screening performed among more than 90 fungalspecies to select the most efficient decolorizer of a lignin-containing paper-industry effluent; a laccase enzyme is sug-gested to be involved in this process (Calvo et al. 1998).Moreover, another strain of C. gallica has recently been re-ported to be an effective pollutant degrader (Pickard et al.1999). To date, we have evidence of only one genomic se-quence for a laccase gene in C. gallica, even though thereare reports of gene families for laccase in some white-rotfungi (Yaver and Golightly 1996; Mansur et al. 1997;Palmieri et al. 2000), and in spite of our numerous attemptsto find more laccase sequences in this fungus (Zapico 1999).This laccase gene (cglcc1) has been cloned and sequenced(GenBank accession No.: AY017340).

Different types of industrial effluents (paper industry, dis-tillery, beer factory, and oil mill wastewaters) have beenshown to increase laccase activity in various white-rot fungi(Mansur et al. 1997; Calvo et al. 1998; Pérez et al. 1998;González et al. 2000; Tsioulpas et al. 2002). All of theseeffluents derive from industries that use plants as a raw ma-terial, and the presence of tannic compounds has been re-ported in some of them (Maestro-Durán et al. 1993). Afterlignin, tannins are the most abundant group of plantpolyphenols and share with lignin a series of common struc-tural polyphenolic features (William et al. 1986). Tannicacid (TA) is the tannin most widely distributed in nature.The possible role of TA as a laccase inductor in C. gallicawas studied from a physiological and molecular point ofview, and the results are presented in this work.

Materials and methods

ChemicalsTA (tannic acid powder pure, United States Pharmaco-

poeia (USP) empirical formula C76H52O46), was obtainedfrom Merck (Darmstadt, Germany). 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was purchasedfrom Boehringer Mannheim (Mannheim, Germany) and2,6-dimethoxyphenol from Fluka (Buch, Switzerland).All other chemicals were reagent grade obtained fromMerck, Boehringer Mannheim, or Sigma–Aldrich Corp.,St. Louis, Mo.

Organism and maintenanceCoriolopsis gallica A-241 was obtained from the IJFM

(Instituto Jaime Ferrán de Microbiología) collection. Thefungal culture was maintained on malt agar slants (2% maltextract, 2% Bacto agar, Difco, Detroit, Mich.), grown for10 days at 28°C and stored at 4°C.

Culture conditionsThe fungus was grown on agar plates with modified

Czapek’s medium (Guillén et al. 1990) for 7 days at 28°C.Ten plugs (1 cm2) were cut and inoculated under sterile con-ditions into 500-mL culture flasks containing 300 mL ofKirk growth medium (nitrogen-limited defined medium)(Kirk et al. 1986). After incubation for 48 h at 28°C in anorbital shaker (200 rpm), 10 mL of the culture medium con-taining little fragments of fungal mycelia was used to inocu-late 250-mL Erlenmeyer flasks containing 90 mL of Kirkmedium. Samples were incubated at 28°C and 125 rpm for7 days. After this time, a filter-sterilized (0.22 µm) solutionof 50 mM TA in distilled water was added to the culture me-dium to reach a final concentration of 50, 100, or 200 µM.Controls without TA were also grown. In all cases, the finalpH of the culture media was 4.6 ± 0.1, which is optimum forC. gallica growth (Calvo et al. 1998). Laccase activity wasmeasured in the extracellular fluid throughout the following9 days of incubation.

Enzymatic activities assaysLaccase activity was determined in the extracellular fluid

of fungal cultures by the method of Wolfenden and Willson(1982), using ABTS as the substrate. Lignin and manganeseperoxidases were measured as described by Tien and Kirk(1984) and Paszczy�ski et al. (1988), respectively. One unitof enzymatic activity is defined as the formation of 1 µmolof product per min.

Zymograms of fungal laccase activityPolyacrylamide gel electrophoresis was performed at alka-

line pH under nondenaturating conditions using a Mini-Protean (Bio-Rad Laboratories, Hercules, Calif.) electropho-resis cell. The separating gel contained 12% acrylamide, andthe buffer solution was 375 mM Tris–HCl (pH 8.8). Thestacking gel contained 5% acrylamide, and the buffer solu-tion was 125 mM Tris–HCl (pH 6.8). The electrode buffersolution contained 25 mM Tris–HCl and 122 mM glycine(pH 8.8). Prestained molecular weight standards (Bio-RadLaboratories) were used. All attempts to determine total pro-teins in the extracellular medium were inaccurate because of

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the interference caused by TA. This problem was overcomeby loading the same amount (25 µL) of extracellular fluidfrom induced and control cultures. To detect laccase activ-ity, the gels were previously equilibrated in 100 mM acetatebuffer at pH 5.0 and stained using 10 mM 2,6-dimethoxy-phenol as the substrate.

RNA preparationsTotal RNA was prepared using the Fast RNA Kit following

the manufacturer’s instructions (BIO 101 Inc., San Diego, Ca-lif., U.S.A.) from fresh C. gallica mycelium collected at day 5of incubation with different concentrations of TA and fromcontrols without TA.

PCRPCR was performed to obtain the hybridization probes for

cglcc1 and gpd1 (glyceraldehyde-3-phosphate-dehydrogenasegene from C gallica). Taq DNA polymerase (Perkin ElmerCorp., Norwalk, Conn.) was used as recommended by themanufacturer, using a Rapidcycler (Idaho Technology, IdahoFalls, Idaho, U.S.A.) thermocycler with a PCR temperatureprogram of 95°C for 1 min, followed by 30 cycles of 95°Cfor 40 s, 55°C for 40 s, 72°C for 1 min, and a final extensionat 72°C for 8 min. All the primers were used at a final con-centration of 0.4 µM.

The hybridization probe for cglcc1 was obtained using as atemplate the plasmid pYES1, which contains the full lengthcDNA of the C. gallica laccase gene (GenBank acc. No.AF263467). The primers 5RT (5′-GCGATTGGCCCCAAGA-CTG-3′) and 3RT (5′-CAGTGGCTGCGTGTTCACAC-3′)were designed to anneal at specific sites of this gene, ampli-fying an intragenic region of 690 bp.

The probe to detect gpd1 transcripts, used as a signal ofconstitutive expression, was obtained using primers DIM (5′-TCAACGGTTTCGGTCGTATT-3′) and RM2 (5′-GTGG-ACGGTGGTCATGAGAC-3′) that amplify a highly con-served fragment of 515 bp of the gpd1 gene from C. gallica(GenBank acc. No. AF297874). The template DNA was theplasmid pHJC5 that included a partial gpd1 cDNA, synthe-sized previously by RT-PCR and cloned on pGEM-T(Promega, Madison, Wisc.).

Labeling of probes was performed using the PCR-amplified fragments of cglcc1 and gpd1 that were run on a1% agarose gel and eluted with the UltraClean-15 DNA pu-rification kit (MO BIO, Solana Beach, California). One mi-crogram of each purified fragment was diluted in H2O to atotal volume of 16 µL, then the DNA samples weredenatured in a boiling water bath for 10 min and chilled onice. After that, 4 mL of digoxigenin (DIG) High Prime(Boehringer Mannheim) were added, mixed, and spinneddown briefly. The reaction was then incubated at 37°C over-night and stopped with 2 µL of 0.2 M EDTA.

Northern analysisThe expression of cglcc1 and gpd1 genes was estimated

using the hybridization probes described above. Approxi-mately 10 µg of total RNA were electrophoresed overnightat 1.2 V/cm in a 1.2% agarose–formaldehyde gel with40 mM MOPS (morpholinepropanesulfonic acid) – 10 mMsodium acetate (pH 7.0) – 1 mM EDTA. RNA band intensi-ties were densitometrically measured after ethidium bromide

staining, and the RNA was then blotted by capillary transferto a Hybond-N membrane (Amersham Biosciences Corp.,Piscataway, N.J.) in 20× SSPE (0.36 M NaCl, 20 mMNaH2PO4, 2 mM EDTA, pH 7.7). The membrane was thenhybridized with the homologous DIG-labeled probes underhigh stringency conditions, following the manufacturer’s in-structions (Boehringer Mannheim). Hybridization signalsand RNA bands were quantified by densitometry usingImagequant Software (Molecular Dynamics, Sunnyvale, Ca-lif.). The transcript levels of the cglcc1 gene were calculatedby dividing their hybridization signal percentages by thecorresponding gpd1 constitutive signals.

Experimental reproducibilityAll the experiments were performed three times. The stan-

dard deviation in all the analytical assays was always lessthan 5%.

Results

Influence of different concentrations of TA on laccaseproduction: spectrophotometric time course

A time course experiment for laccase activity in theextracellular fluid of C. gallica showed that it was clearly in-fluenced by the addition of different concentrations of TA(Fig. 1). At day 2 of fungal growth in the presence of 50 µMTA, laccase values were higher than those of the controlsand reached a maximum after 4 days. From day 4 to 9 of in-cubation with 50 µM TA, the activity decreased, althoughat day 9, it was still higher than controls. At a final concen-tration of 100 µM TA, the increase of laccase activity wasmuch higher than that detected at 50 µM but occurred later,reaching a maximum after 7 days following TA addition.The highest TA concentration assayed (200 µM) caused apartial inhibition of C. gallica growth. The same observa-tion regarding the negative effect of tannic compounds onthe growth of several filamentous fungi has been reported(Davidson et al. 1938; Nobles 1948; Scalbert 1991) and for

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Carbajo et al. 1043

Fig. 1. Time course for laccase activity monitored in theextracellular medium of Coriolopsis gallica grown in the absence(�) and in the presence of 50 µM (�) and 100 µM (�) tannicacid. Time is expressed in days after the addition of tannic acid.Experimental data are the means of three experiments, and theexperimental error was never greater than 5%.



this reason, the results of laccase activity at this TA concen-tration were considered not to be comparable.

In all cases, except in controls without TA, an increase ofbrown color in the extracellular fluid was observed duringthe first days after the addition of TA (data not shown), atwhich time laccase activity also started to be detected(Fig. 1). Moreover, the color intensity was directly corre-lated with TA concentration in the cultures containing 50and 100 µM of TA. This color became less intense as thelength of incubation increased, along with increasing valuesof laccase activity in the extracellular fluid.

Lignin peroxidase and manganese peroxidase were not de-tected in the extracellular fluid of C. gallica grown with dif-ferent concentrations of TA nor in the controls without TA.

Influence of different concentrations of TA on laccaseproduction: zymograms

The results of zymograms of laccase activity from day 1to 9 after the addition of various concentrations of TA areshown in Fig. 2. The increase in the laccase activity detectedin the fungal culture containing 50 and 100 µM TA (Fig. 1)was also observed in the zymograms (Fig. 2). As in thespectrophotometric assays, the effect of TA on increasedlaccase activity was observed earlier in the medium contain-ing 50 µM TA than in the culture with 100 µM TA (Fig. 2).In the last 5 days of incubation, the induction of laccase pro-duction became stronger in the cultures containing 100 µMTA, whereas a decrease in laccase signals were observed inthe presence of 50 µM TA, although levels were still higherthan controls at day 9. In addition, the density of the inducedenzymes bands was greater than those of controls withoutTA. Moreover, the bands of laccase activity in the controlsshowed a sharp pattern during the first 4 days assayed, be-coming more diffuse towards the latter part of the experi-ment (Fig. 2, lanes b).

Transcription analysis of cglcc1The possible correlation between the increase in laccase

activity observed in culture media supplemented with TAand the levels of cglcc1 transcripts was analyzed by North-ern blotting. RNA was prepared from C. gallica myceliumharvested on day 5 after the addition of 50, 100, and 200 µMof TA and from controls. Good quality RNA was obtainedfrom controls and from samples with 50 and 100 µM, but allthe attempts to extract RNA from mycelium incubated in thepresence of 200 µM TA were unsuccessful. The interferenceof tannic compounds in the extraction of RNA has previ-ously been reported in the case of polyphenolic-rich materi-als (John 1992). In the same way, TA also causes aninaccurate spectrophotometric quantification of RNA; thisproblem was overcome by using the hybridization signal ofgpd1 from C. gallica as a loading control.

The effect of TA on cglcc1 expression is shown in Fig. 3,which also shows the ratio between the hybridization signalsof cglcc1 and gpd1 (Fig. 3D). In the range of TA concentra-tions tested, the results showed the existence of a clear cor-relation between TA concentration and induction of cglcc1expression. Our results showed that relative levels of cglcc1transcripts in cultures containing 50 and 100 µM TA werehigher than controls without TA, in agreement with thelaccase activity determined spectrophotometrically and thezymogram data obtained on day 5. This observation indi-cates that TA can have an important effect on the inductionof cglcc1 gene transcription, suggesting that these transcriptsare translated to an active laccase protein.

Discussion

Until now, little work has been done regarding the regula-tion of laccase gene expression in white-rot fungi (Eggert etal. 1996; Collins and Dobson 1997; Mansur et al. 1998;Palmieri et al. 2000). The addition of inducers is one of thesimplest methods to increase the yield of enzyme produc-tion. It has been shown that tannic compounds are able to in-crease laccase activity in several basidiomycetes (Pickardand Westlake 1970; Arora and Sandhu 1984) and ascomy-cetes (Kim et al. 1995), although, as far as we know, the mo-lecular basis of such induction has not been reported. Theresults presented here demonstrate that the expression of thelaccase gene cglcc1 of C. gallica is transcriptionally induci-ble by TA, giving rise to an enhancement of laccase activ-ity. The induction of laccase in several fungi by a numberof low molecular weight phenolic compounds (Koroljova-Skorobogat’ko et al. 1998), aromatic acids (Farnet et al.1999), a variety of flavonoids (Pickard and Westlake 1970),and different lignin preparations (Arora and Sandhu 1984)has been demonstrated at the physiological level. Our resultsshowed that TA can be considered as an efficient inducer oflaccase production by C. gallica, as revealed by the increaseof enzymatic activity detected and, as will be discussed later,by the enhancement of the laccase gene at the transcriptionlevel.

An increased level of laccase activity in C. gallica andother white-rot fungi growing in the presence of diversetypes of industrial wastewaters has been frequently detected(Ardon et al. 1998; Pérez et al. 1998; Calvo et al. 1998;

© 2002 NRC Canada


Fig. 2. Zymograms of laccase activity monitored in theextracellular fluid of Coriolopsis gallica grown in the absence(lanes b) and in the presence of 50 µM (lanes c) and 100 µM(lanes d) tannic acid at different days of incubation after the ad-dition of tannic acid. Molecular weight standards are representedby lanes a.



González et al. 2000; Yagüe et al. 2000; Tsioulpas et al.2002), and the presence of tannins has been described insome of them (Maestro-Durán et al. 1993; Yagüe et al.2000). Our results suggest that laccase induction producedby this type of industrial effluents could be ascribed, in part,to TA or to structurally related molecules.

A detoxification of toxic phenols by means of polymeriza-tion reactions has been suggested as one of the physiologicalroles of fungal laccases (Bollag et al. 1988; Thurston 1994).Our observation that an increase of color in extracellular fluidof cultures containing TA coincided with detection of laccasesupports the proposed role of the enzyme in the detoxificationof phenolic compounds by polymerization reactions. In addi-tion, the fact that this color became less intense with time,along with increasing values of laccase activity, suggests theinvolvement of laccase in the further degradation of thesecompounds (Bollag et al. 1988) without disregarding the pos-sible contribution of other enzymes. Studies using differentbasidiomycetous strains are being carried out in our labora-tory to obtain further information on this subject.

Laccase induction by TA could be indicative of an envi-ronmental switching response developed by fungi to oxidizeand, therefore, to decrease the potentially toxic effect of TAand related compounds. Recently, some of our results re-garding the decolorization of tannin-rich wastewaters alsosuggested a toxic effect of polyphenols on the growth of C.gallica and a further adaptation of the fungus along with apolymerization of phenol compounds (Yagüe et al. 2000).Similarly, an induction of laccase by other potential toxiccompounds in connection with an adaptative fungal responseto diminish their toxic effect has also been suggested for2,5-xylidine in Pycnoporus cinnabarinus (Eggert et al.1996), 2,5-xylidine and 1-hydroxybenzotriazole in Trametesversicolor (Collins and Dobson 1997), and for undefined

compounds present in distillery vinasses in Trametes sp. I-62 (Mansur et al. 1997).

At the transcriptional level, induction of laccase in theascomycete Neurospora crassa has been well characterized(Linden et al. 1991; Tamaru et al. 1994). Moreover,Schouten and coworkers (2002) have recently described theinduction of transcripts for the laccase gene bclcc2 from theascomycete Botrytis cinerea when a solution of tannic acidwas added to the culture medium. In contrast, in basidio-mycetous fungi, studies regarding induction of laccase tran-scription are still scarce (Eggert et al. 1996; Collins andDobson 1997; Mansur et al. 1998; Palmieri et al. 2000). Ourresults showed a direct relation between an increase incglcc1 transcript levels and the enhancement of laccase ac-tivity values produced by C. gallica in the presence of TA.

The presence of various laccase isozymes in the extra-cellular fluid of C. gallica growing in TA is suggested by thethick shape of the bands attained in the zymograms pre-sented in this study. Laccase isozymes can be ascribed to alaccase gene family or to postranslational modifications of asingle protein. The latter could be the case for cglcc1, giventhat several potential places of glycosylation can be found inthe putative amino acidic sequence deduced from this gene.Glycosylation is a common feature that has been demon-strated in many other fungal laccase proteins from basidio-mycetes (Perry et al. 1993; Giardina et al. 1996). Theincrease of laccase activity could be, among others, the re-sult of several aspects: (i) an increased production of laccasemRNA, (ii) an increased stability of laccase mRNA tran-scripts, (iii) increased production of the active protein, and(iv) an increased half-life of laccase protein. Work is cur-rently underway to examine some of these possibilities.

The induction of laccase production in different filamen-tous fungi seems to be specific for certain aromatic

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Carbajo et al. 1045

Fig. 3. (A) Total RNA samples from mycelium of Coriolopsis gallica harvested on day 5 after the addition of 0, 50, and 100 mM ofTA (lanes 1, 2, and 3, respectively). Northern blot analysis of cglcc1 gene expression (B) and gpd1 gene transcripts (C). (D) cglcc1transcript levels represented as the ratio between the hybridization signals of cglcc1 and gpd1.



compounds; therefore, some precise mechanisms of transcrip-tional activation are likely to be involved. We have found aputative xenobiotic response element (XRE) in the promoterof cglcc1 of C. gallica (GenBank acc. No. AY017340). Thesequence GTGCCAT, reported by Fujisawa-Sehara et al.(1988) (coincidences with the XRE consensus sequence areunderlined), is present 115 bp upstream of the TATA box.The presence of these putative XREs has been reported inseveral white-rot fungal lcc promoters (Coll et al. 1993;Giardina et al. 1995; Collins and Dobson 1997; Mansur etal. 1997), suggesting that transcription of laccase genes maybe activated by stressing compounds, such as TA.

The high degree of toxicity for human and animal healthshown by some synthetic laccase inducers makes their userather limited as potential enhancers of laccase productionon an industrial scale in the future. TA is one of the first re-ported molecules of natural origin that is able to increaselaccase levels, making its use, in principle, more suitablefrom an industrial standpoint. In addition, the synthetic ori-gin of some of the inducers studied to date make it difficultto elucidate their role in nature. In contrast, tannic com-pounds are natural molecules widely distributed in the envi-ronment, mainly as components of the external surfaces ofplants, which represent the first defense mechanism againstexternal aggressions. The results presented here permit us topropose an essential role of tannins as inducers of fungallaccases in the initial steps of wood degradation in naturalsystems.

Acknowledgements

We are grateful to G. del Solar, M. Espinosa, and A.D.W.Dobson for their critical reading of the manuscript. Researchwas financed by Comisión Interministerial de Ciencia yTecnología (CICYT, Madrid, Spain) BIO 97-0655-E. H.Junca acknowledges financial support from G. Díaz deJunca. T. González and E. Zapico acknowledge support froma Mutis Programme doctoral grant from Agencia Españolade Cooperación Internacional (AECI) (Spain). S. Yagüe ac-knowledges a grant from Ministerio de Ciencia y Tecno-logia. M.C. Terrón acknowledges a postdoctoral grant fromConserjería de Educación y Cultura de la ComunidadAutónoma de Madrid (Spain).

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