Use of Multiplex Reverse Transcription-PCR To Study the Expression of a Laccase Gene Family in a Basidiomycetous Fungus

Abstract

Laccases produced by white rot fungi are involved in the degradation of lignin and a broad diversity of other natural and synthetic molecules, having a great potential for biotechnological applications. They are frequently encoded by gene families, as in the basidiomycete Trametes sp. strain I-62, from which the lcc1, lcc2, and lcc3 laccase genes have been cloned and sequenced. A multiplex reverse transcription-PCR method to simultaneously study the expression of these genes was developed in this study. The assay proved to be quick, simple, highly sensitive, and reproducible and is particularly valuable when numerous samples are to be analyzed and/or if the amount of initial mRNA is limited. It was used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and 3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in Trametes sp. strain I-62. These aromatic compounds produced different induction patterns despite their chemical similarity. We found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase activity while also producing the highest increase in gene expression; 3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript amounts of each gene fluctuated dramatically in the presence of these three inducers, while the total amounts of laccase mRNAs seemed to be modulated by a coordinated regulation of the different genes.

White rot basidiomycetes are a group of organisms able to completely degrade lignin. They play an important role as recyclers of this abundant polymer actively synthesized by plants in the biosphere. Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2) are glycosylated polyphenoloxidases which constitute an essential part of a complex nonspecific ligninolytic enzymatic system secreted by these fungi (26, 32, 43). These enzymes are also attracting increasing interest due to their capacity to degrade a broad diversity of natural and synthetic materials, with potential industrial applications such as upgrading of animal feed (1, 21), pulp and paper production, textile dye bleaching, bioremediation and effluent detoxification, use as washing powder components, removal of phenolics from wines, and transformation of antibiotics and steroids (5).

Laccases are typically produced by white rot fungi as multiple isoenzymes (3, 10, 26, 32). Such diversity in laccase isoenzymes was first attributed to posttranslational modifications of the same gene product, but the characterization of several laccase gene families (13, 14, 15, 23, 30, 33, 35, 37, 42, 44, 46, 47) suggested that at least a part of this biochemical diversity could be the result of the multiplicity of laccase gene in fungal genomes. Extracellular laccases are constitutively produced in small amounts in several fungi (3, 4, 10, 31), but the production of these enzymes can be considerably enhanced by a wide variety of substances such as different aromatic compounds. However, there are not many reports in the literature regarding laccase regulation at the transcriptional level. The study of laccase gene expression by traditional methods such as Northern blot analysis is difficult for fungi that have a family of these genes because the homology between genes of a same family complicates the selection of specific probes. Reverse transcription coupled to the PCR technique (RT-PCR) has been used to quantitatively study the expression of laccase genes under different environmental conditions (9, 40, 42, 48). RT-PCR has several advantages such as simplicity, rapidity, and high sensitivity, but the reliability of this technique as a quantitative method is controversial. Nevertheless, the inherent quantitative capacity of RT-PCR has been demonstrated (20), and its pitfalls and potentials as a powerful tool for analyzing RNA have been reviewed by Freeman et al. (11). Multiplex PCR is a variant of PCR in which two or more loci are simultaneously amplified in the same reaction (7). For fungi having several laccase genes, the use of multiplex RT-PCR assay could facilitate the study of their differential expression under different culture conditions. The white rot fungus Trametes sp. strain I-62 (CECT 20197) is a strain with a great potential for biotechnological applications. The high detoxification capacity displayed by this fungus on distillery effluents and the possible role of laccases in this process have been studied in our laboratory (16), and Mansur et al. (30, 31) have described a family of three laccase genes in this strain that are differentially regulated. They demonstrated that veratryl alcohol increased the expression of the lcc1 and lcc2 Trametes sp. strain I-62 laccase genes. The capacity of aromatic compounds to induce laccase activity is strongly related to their chemical structure (41); we therefore thought it would be interesting to study the effect of subtle changes in these types of molecules, such as the positions of the substituents groups on the aromatic ring, on their inductive effect on laccase activity. Trametes sp. strain I-62 has been used in this work as a model system to investigate aromatic molecules acting as inducers of laccase transcription in fungi and for the study of their effects on differential laccase gene expression.

In the present work a multiplex RT-PCR method to study laccase gene expression in the basidiomycete Trametes sp. strain I-62 has been designed and optimized. We demonstrate the reliability and simplicity of this assay through an applied comparative study to determine the effect of veratryl alcohol and of its 2,5-dimethoxibenzyl alcohol and 3,5-dimethoxibenzyl alcohol isomers on the differential expression of the three laccase genes cloned from this fungus.

MATERIALS AND METHODS

Organism and culture conditions.

The basidiomycete Trametes sp. strain I-62 was isolated from decayed wood in Pinar del Río, Cuba (30). The fungal culture was maintained on agar plates with modified Czapeck medium (18). Cultures grown for seven days at 28°C were stored at 4°C. Submerged cultures were prepared by the inoculation of eight 1-cm² plugs from these plates under sterile conditions into 500-ml Erlenmeyer flasks containing 300 ml of the same growth medium and four 1.5-cm-diameter glass beads. After incubation for 24 h at 28°C in an orbital shaker (100 rpm), 7.5-ml inocula were transferred into 250-ml flasks containing 75 ml (total volume) of Kirk medium (24) without the addition of veratryl alcohol. The effect of filter-sterilized veratryl alcohol (3,4-dimethoxybenzyl alcohol [3,4-DMBA]), 2,5-dimethoxybenzyl alcohol (2,5-DMBA), and 3,5-dimethoxybenzyl alcohol (3,5-DMBA) isomers was studied by two different strategies: (i) adding each compound to the culture medium, from the beginning of the incubation time, at a final concentration of 0.37 mM and measuring laccase activity daily for 8 days and (ii) monitoring this enzymatic activity over a 43-h period following the addition of these aromatic compounds (0.37 mM) to 8-day-old cultures of Trametes sp. strain I-62 grown in Kirk medium without 3,4-DMBA. All experiments were run in triplicate.

Laccase activity.

Laccase activity was determined by taking 1-ml samples of the extracellular fluid of fungal cultures (45) with ABTS (2,2′-azinobis-3-ethylbenzthiazoline-6-sulfonate) as the substrate. One unit of laccase activity is defined as the formation of 1 μmol of oxidized ABTS per min.

Gravimetric analysis.

To compare the growth of Trametes sp. strain I-62 in the liquid cultures in the presence of the different aromatic compounds, the mycelium was harvested, washed with sterile H₂O, frozen at −70°C, and freeze-dried to determine the dry weight of each sample.

Total-RNA preparation.

To study the effect of the three aromatic compounds mentioned above on lcc gene transcription, fresh mycelium samples (approximately 10 mg) were harvested at different time points (7, 19, 31, and 43 h) following the addition of these compounds to the 8-day-old fungal cultures. RNA extraction was performed by using the Fast RNA kit-Red, as specified by the manufacturer (BIO 101, Inc., La Jolla, Calif.). The total RNA concentration was determined spectrophotometrically. To remove contaminating DNA, 1 U of RQ1 DNase (Promega) per μg of RNA was added to each RNA sample and the samples were incubated for 30 min at 37°C. The RNA was phenol-chloroform extracted, precipitated with isopropanol, washed with 70% ethanol, and dissolved in sterile water. The integrity of the RNA was verified by electrophoresis on 0.8% agarose gels followed by ethidium bromide staining.

cDNA synthesis.

First-strand cDNA synthesis was carried out using 2 μg of total RNA as template and the cDNA synthesis kit from Roche (used as specified by the manufacturer).

Multiplex PCR amplification.

A general scheme of the multiplex PCR method designed in this work is illustrated in Fig. 1A. A preliminary study to select the optimal PCR conditions to amplify three fragments (corresponding to the lcc1, lcc2, and lcc3, laccase genes) with the same efficiency was performed. A careful primer selection for multiplex PCR application was done, assessing critical factors such as compatibility, in terms of not producing any additional bands or spurious hybridizations of primer pairs to each other in amplification reactions. The primer sequences are showed in Fig. 1A. cDNAs corresponding to each lcc gene, cloned in the pGEM-T vector (Promega), were used as templates. They had been previously synthesized and cloned by González (17).

FIG. 1.

(A) Binding sites and sequences of the primers used in the multiplex PCR reactions for the simultaneous amplification of the lcc1, lcc2, and lcc3 laccase genes from Trametes sp. strain I-62. Genes are represented under the scale, and dark regions indicate introns. Arrows show primer binding sites. The PCR products obtained from genomic DNA and from cDNA amplification are represented. The DEN1 and RM2 primers are specific for gpd1 amplification and produce the same 500-bp PCR product when the amplification is from genomic DNA or from cDNA (data not shown). (B) Amplification of lcc1, lcc2, and lcc3 laccase gene fragments by multiplex PCR. PCR products derived from genomic DNA or from cDNA amplification are distinguished by their size in agarose gel electrophoresis (1% agarose). Lane 1, characteristic bands from genomic DNA amplification using the three pairs of primers simultaneously in the same PCR mixture are lcc1, 1,010 bp; lcc2, 853 bp; lcc3, 565 bp. Lane 2, amplification from cDNAs (equimolar amounts of each cDNA target template): lcc1, 675 bp; lcc2, 550 bp; lcc3, 433 bp. Lanes 3 to 5, the same reactions using each cDNA template separately (3, lcc1; 4, lcc2; 5, lcc3), and the three pair of primers simultaneously, prove that they are highly specific and do not interact to produce additional bands other than those expected. MX, molecular weight marker.

PCR mixtures were prepared by adding equimolar amounts of the three laccase cDNA genes and their corresponding pairs of primers. Reactions with each single template were performed. Then 3.2-fold (0.5-log-unit) serial dilutions of 1 pg of lcc1, lcc2, and lcc3 templates were prepared to calculate the amplification efficiency of each gene in the corresponding PCR amplifications. The same procedure was performed to amplify a fragment of the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd1; GenBank accession no. AF 297874), which was further used as a control to normalize differences in the total RNA input or in the RT reaction efficiencies.

PCR amplifications were performed in a Rapidcycler (Idaho Technology) thermocycler. cDNA templates were mixed with primers and Taq polymerase (Pelkin-Elmer) in a solution containing the standard components of a PCR DNA amplification reaction (38). Different parameters were adjusted to obtain maximal specificity and comparable high PCR product yields for the three individual laccase genes. The MgCl₂ concentration in the reaction mix was increased from 2 to 4 mM in 0.5 mM steps; the annealing temperature was tested in the range of 53 to 61°C in 2°C steps, and 20 to 30 PCR cycles were tested in 5-cycle steps. All other parameters remained unchanged unless otherwise indicated. The basic PCR program was an initial denaturation step at 95°C for 45 s, 30 s at the annealing temperature, and 72°C for 2 min for the appropriate number of cycles, one final extension step at 72°C for 7 min followed by a step at 4°C until further storage of reactions at −20°C.

Quantitative and statistical analysis.

For each condition assayed, three independent amplification reactions were done. PCR products (10 μl for each reaction) were separated by agarose gel electrophoresis (1.5% agarose) and visualized after staining for 10 min in a 1-μg/ml ethidium bromide solution. Densitometric analysis of Polaroid film gel images was performed using Image Quant 3.3 software (Molecular Dynamics). Standard curves were generated by plotting the replicated PCR product yield (i.e., the intensity of ethidium bromide staining) as a function of the initial concentration (as log dilution⁻¹). The regression-line equations and correlation coefficients were calculated to P <0.001.

Optimal conditions for multiplex PCR of the three laccase genes and for amplification of the gpd1 fragment were used to study the expression of these genes after the addition of 3,4-DMBA, 2,5-DMBA, and 3,5-DMBA. Two replicate PCR amplifications were run to amplify cDNAs from 5 μl of each cDNA synthesis reaction.

Levels of lcc mRNAs were expressed in arbitrary units, as the ratio between lcc transcript levels (previously normalized according to size differences) and those of gpd1 calculated by the following equation: laccase/(gpd1_sample/gpd1_average). For all experiments and determinations, variability coefficients between independently replicated samples were calculated. Statistical differences were determined by the t test for mean comparison (with P <0.001).

RESULTS

Multiplex RT-PCR method designed for the comparative analysis of laccase lcc1, lcc2, and lcc3 mRNA levels.

The basis of the multiplex PCR assay designed for the comparative and simultaneous study of transcript levels of the three laccase genes from Trametes sp. strain I-62 are illustrated in Fig. 1. Because of the extreme sensitivity of this assay, even minute amounts of contamination by genomic DNA can lead to aberrant results in the quantitative RT-PCR amplifications. Primers were designed in such a way that the products amplified from genomic DNA can be distinguished, by agarose gel electrophoresis, from those obtained by cDNA target amplification. This is done by using primers that amplify DNA regions comprising various introns (Fig. 1A) and permits the detection of any remaining contaminating DNA in the RNA samples for the expression studies.

Two other prerequisites were essential to make this method reliable and functional: each set of primers must be highly specific to amplify only its corresponding target, and they should not interact or produce any additional bands than those expected in the multiplex PCR amplifications. These requirements were tested for the amplification of each single cDNA target by adding, along with the corresponding set of primers, those of the other two lcc genes together in the same reaction to be sure that each set of primers anneals only with their respective cDNA template and that they give rise only to the expected unique PCR product (Fig. 1B).

PCR conditions were adjusted to amplify exclusively the expected products. Nevertheless, the challenge in developing a multiplex PCR assay is in optimizing the reaction in such a way that all targets are amplified at a similar efficiency (25). Taking into account this additional prerequisite, adjustments to the PCR conditions were aimed at not only ensuring specificity but also obtaining the same amplification efficiency for the products of the three laccase genes. This was achieved by adjustments of MgCl₂ concentration, annealing temperature, and number of PCR cycles. The amplification of lcc1, lcc2, and lcc3 cDNA fragments, at concentrations from 1 to 0.003 pg of template, proceeded with the same efficiency during 30 PCR cycles using 2.5 mM MgCl₂ and at an optimum annealing temperature of 59°C (Fig. 2A). These optimizations are represented by overlapping of the regression lines calculated from the relationship between the PCR product yield and the template input in each three reactions shown in Fig. 2C.

FIG. 2.

Demonstration of equal amplification efficiency of the fragments corresponding to lcc1 (black circle), lcc2 (black square), lcc3 (black triangle), and gpd1 (black diamond) cDNAs from Trametes sp. strain I-62. (A and B) PCR products of 30 and 25 amplification cycles from 3.2-fold (0.5-log) serial dilutions of lcc1, lcc2, and lcc3; and gpd1 templates, respectively, as seen on ethidium bromide-stained 1.5% agarose gels. (C and D) Regression analysis to determine the dependence of PCR product yield (measured by densitometry) on template input in each reaction. Each data point represents the mean obtained from three replicate PCRs.

The PCR amplifications were moreover adjusted to amplify also the gpd1 fragment used as internal control (Fig. 2B). The amplification was done in a separate PCR mixture, prepared in each case from the same RT product, to normalize differences in total RNA target input and quality and in the RT efficiency. The optimal MgCl₂ concentration was 2.5 mM, and the optimal annealing temperature was 53°C. The number of PCR cycles was reduced to 25, taking into account that gpd1 is a multicopy gene which is expressed at much higher levels than the mRNA being studied, so that the PCR plateau will be achieved before that of laccase genes. This was confirmed by performing amplifications from several dilutions of the RT reaction product as target (data not shown). Figure 2D shows that the slope of the regression line, calculated for the analysis of gpd1 amplification at the same range of target concentration, is very similar to those of lcc1, lcc2, and lcc3. Table 1 shows data from the several multiplex PCR quantifications. The reproducibility of the assays should be noted.

TABLE 1.

Reproducibility of multiplex PCR quantifications^a

Template input (pg)	Target	PCR product yield (IQ units)b	Coefficient of variation (%)
1	lcc1	28.6, 25.6, 27.8	5.7
	lcc2	23.0, 22.9, 25.0	5.0
	lcc3	18.3, 20.0, 25.0	7.8
	gpd1	21.1, 20.0, 20.8	2.8
0.312	lcc1	20.0, 18.6, 19.9	4.0
	lcc2	18.6, 17.5, 19.1	4.4
	lcc3	13.3, 11.8, 13.1	6.5
	gpd1	18.5, 16.9, 19.0	6.1
0.097	lcc1	16.0, 15.5, 15.8	1.6
	lcc2	11.0, 10.8, 11.5	3.2
	lcc3	10.4, 11.2, 12.3	8.4
	gpd1	13.9, 14.0, 13.3	2.8
0.030	lcc1	12.0, 11.6, 10.9	4.8
	lcc2	8.3, 7.4, 8.5	7.2
	lcc3	7.3, 8.6, 7.6	8.6
	gpd1	12.2, 11.9, 13.0	4.6
0.009	lcc1	3.0, 2.9, 2.8	3.4
	lcc2	3.0, 2.7, 2.6	7.4
	lcc3	2.8, 2.5, 2.9	7.7
	gpd1	6.5, 6.1, 5.8	6.1
0.003	lcc1	2.3, 3.2, 2.9	8.9
	lcc2	3.0, 2.6, 3.0	8.0
	lcc3	2.2, 2.6, 2.3	8.7
	gpd1	2.3, 2.8, 2.5	10.0

^aThe results of three independently replicated samples for each gene at five different concentrations are shown.

^bIQ units are the arbitrary units of the ImageQuant software used for densitometry quantification of the PCR products.

The different signals we obtained by RT-PCR following the expression of gpd1 in Trametes sp. strain I-62 were independent of variables such as culture age or aromatic compound added, following the rules of a housekeeping gene, which are expressed at constitutive, high, and stable levels. It is worth pointing out that all the above results were obtained after standardization of conditions for the multiplex RT-PCR method that guaranteed an optimal amplification.

Laccase activity in cultures with 3,4-DMBA and its 2,5-DMBA and 3,5-DMBA isomers.

Once the multiplex RT-PCR method for the comparative analysis of lcc transcripts had been standardized, the laccase activity and growth of the fungus in cultures supplemented with veratryl alcohol (3,4-DMBA) and its isomers 2,5-DMBA and 3,5-DMBA were analyzed. These compounds have an identical chemical composition and differ only in the distribution of the groups in their aromatic rings (Fig. 3C). The three isomers caused an increase in the extracellular levels of the enzyme, which could be detected after day 3 of culture (data not shown). In cultures to which 3,4-DMBA was added, the highest levels of laccase activity were detected on days 4 and 5 of the experiment. These levels were twice as high as those observed in the controls grown in Kirk medium (without any aromatic compound). The highest laccase activity in the presence of 3,5-DMBA occurred on the same day and it was three times higher than those of the controls. Nevertheless, the highest induction was produced by 2,5-DMBA. In this case, the highest laccase activity was detected on day 5 of culture, yielding a value six times higher than that of the control. The same trends were observed when laccase activity was monitored after the addition of each isomer to 8-day-old cultures of Trametes sp. strain I-62 in Kirk medium (Fig. 3A). An increase in extracellular laccase activity was detected 6 h after the addition of 2,5-DMBA and 3,5-DMBA and 11 h after the addition of 3,4-DMBA. The enzymatic activity continued to increase in the presence of the three isomers until the end of the experiment (43 h). At this time, laccase activities attained in the presence of 3,4-DMBA, 3,5-DMBA, or 2,5-DMBA were two, three, and six times higher, respectively, than that of the control.

FIG. 3.

Effect of 3,4-DMBA, 2,5-DMBA, and 3,5-DMBA isomers on laccase activity and lcc gene expression in 8-day-old Trametes sp. strain I-62 submerged cultures. (A) Time course of laccase activity in the extracellular fluid in control Kirk medium (black diamond) and in Kirk medium after the addition of 3,4-DMBA (black triangle), 2,5-DMBA (black circle), and 3,5-DMBA (black square). Each data point represents the mean of three replicate determinations. (B) Effect of the three isomers on the level of lcc1, lcc2, and lcc3 laccase gene transcripts analyzed by multiplex RT-PCR. The amplification of a fragment from gpd1 gene was used as an internal control for each sample. (C) Chemical structures of the 3,4-DMBA, 2,5-DMBA, and 3,5-DMBA isomers. Numbers on the aromatic rings indicate the carbon atoms.

Cultures of Trametes sp. strain I-62 grown for 8 days in Kirk medium supplemented with each of the three isomers showed that these compounds can also affect the growth and morphology of this fungus (data not shown). Curiously, in the presence of 2,5-DMBA, fungal growth decreased and the mycelial pellets were less numerous and smaller than those when the fungus was grown in the control medium without any aromatic compound. In contrast, in media with 3,4-DMBA, the fungal pellets were significantly larger than those of the control. Cultures with 3,5-DMBA showed no differences with respect to controls. All the visually detected differences were confirmed by gravimetric analysis of dry-weight mycelium.

Effect of 3,4-DMBA, 2,5-DMBA and 3,5-DMBA on the temporal expression of lcc genes.

The multiplex RT-PCR method was used to study the effect of the three isomers on lcc transcripts in 8-day-old cultures of Trametes sp. strain I-62. Changes in the relative mRNA levels of lcc1, lcc2, and lcc3 at different times after the addition of inducers are shown in Fig. 3B. It was evident that the three compounds tested increased the laccase transcript levels, but the induction associated with each one was different. Relative levels of lcc mRNAs determined by densitometric quantification of the RT-PCR products, as indicated in Materials and Methods, are shown in Fig. 4. Transcript levels of lcc1, lcc2, and lcc3 decreased in the control during all hours analyzed. They achieved their minimum at the end of the experiment (Fig. 4A). In the presence of 2,5-DMBA, a marked increase in mRNA levels of the three laccase genes was detected in the first sample, that is, 7 h after the addition of this compound (Fig. 4B). At this time the highest levels of lcc1, lcc2, and lcc3 were achieved, but the maximum level of lcc2 expression, which was in fact higher than those of the other two genes, occurred later (31 h after the addition of 2,5-DMBA).

FIG. 4.

Relative levels of Trametes sp. strain I-62 lcc mRNAs determined by densitometric quantification of the RT-PCR products. Each data point represent the mean PCR product yield obtained from two independent amplifications. Arbitrary units express the ratio between the lcc transcript levels (intensities normalized according to PCR product size) and those of gpd1. This ratio is expressed as laccase/(gpd1_sample/gpd1_average). (B to D) The changes in lcc transcripts levels (lcc1 [black diamond], lcc2 [black circle], and lcc3 [black triangle]) are represented at different times after the addition of 2,5-DMBA (B), 3,4-DMBA (C), and 3,5-DMBA (D) to 8-day-old cultures of Trametes sp. I-62 in Kirk medium. (A) Control cultures in Kirk medium without any aromatic compound. (E) Total lcc transcript levels calculated by the addition of the relative levels of lcc1, lcc2, and lcc3 mRNAs in each sample: control (black diamond), 3,4-DMBA (black triangle), 2,5-DMBA (black circle), and 3,5-DMBA (black square).

The rapid and marked induction of laccase gene expression by 2,5-DMBA correlates well with the changes in laccase activity detected in the culture media. Of the three isomers tested, 3,4-DMBA had the smallest effect on the laccase transcript levels (Fig. 4C). In fact, 7 h after its addition to the culture medium, the levels of lcc1 and lcc2 were identical to those of the noninduced control and the levels of lcc3 were even lower. The increase of laccase activity in the culture medium was also slower in the presence of 3,4-DMBA than with the two other compounds (Fig. 3A). At the mRNA level, significant changes were not detected in the time course of lcc gene expression from the beginning of the experiment, and only a slight decrement during the final hour was detected. 3,5-DMBA produced great variations in laccase mRNA levels (Fig. 4D). As with 2,5-DMBA, the induction by 3,5-DMBA was detectable from the first sample, coinciding with the increment of laccase activity in the culture medium, but the lcc1 mRNA levels in the presence of 3,5-DMBA were higher than those achieved with 2,5-DMBA. In addition, the temporal patterns of induction of lcc2 and lcc3 associated with 3,5-DMBA occur in opposite directions, suggesting that the increase in the transcript levels of one gene would be compensated by the reduction in the quantities of the other.

The total transcript levels of lcc genes in each sample were calculated by addition of all the quantified relative levels of lcc1, lcc2, and lcc3 mRNAs (Fig. 4E). Although they were always higher in the presence of 3,5-DMBA than of 3,4-DMBA, the shapes of the curves are very similar to those attained for the time course of extracellular laccase activity (Fig. 3A). The notable inductive effect of 2,5-DMBA on laccase genes seems to occur through fast changes in mRNA levels, always higher than those corresponding to the other isomers tested. Dramatic decreases in lcc transcript levels could be explained as a mechanism which could be used by the organism to avoid an excessive waste of energy while maintaining, higher levels of enzymes possibly playing the role of allowing survival and/or growth of the fungus under stressing conditions such as the presence of aromatic compounds.

DISCUSSION

RT-PCR is the most sensitive technique currently available for mRNA detection and quantification. Because of its extreme sensitivity, this method requires substantial preexperimental planning and design to avoid aberrant results produced by the exponential nature of the amplification reactions. In fact, quantitative application of RT-PCR has been the subject of considerable debate for several years. Different alternatives such as the choice of internal or external RNA standards and quantification strategies (competitive or noncompetitive and kinetic [real-time] amplification) have been developed to solve problems affecting accurate quantification (11). Multiplex PCR is a demanding PCR technique used for genetic screening, microsatellite analysis, and other applications in which amplification of several products in a single reaction is necessary. Multiplex RT-PCR permits semiquantitative analysis of expression pattern and transcript levels of a series of related genes.

The multiplex RT-PCR method developed in the present work allows the relative quantification of laccase transcripts from Trametes sp. strain I-62 in a simple and rapid analysis of gene expression. The assay proved to be highly sensitive, accurately detecting differences in the order of 3.2-fold in template input. The additional amplification of gpd1 used as a control validates the integrity of target mRNA and corrects differences in RNA loading and in RT efficiency. The reproducibility of the assays was confirmed by the values of the coefficient of variation from replicate quantifications, which were always lower than 10% (Table 1). The potential of this method was demonstrated through by studying the effect of three aromatic compounds on lcc1, lcc2, and lcc3 gene expression.

Veratryl alcohol (3,4-DMBA) has previously been reported to be an inducer of the lcc1 and lcc2 genes from Trametes sp. strain I-62 (31). 2,5-DMBA and 3,5-DMBA are isomers of 3,4-DMBA, and they had not been previously studied as laccase inducers. The role of 3,4-DMBA in laccase production has been controversial. It is a secondary metabolite synthesized de novo and secreted by different white rot fungi (2). Its capacity to induce laccase activity differs between organisms: an increase in laccase activity, ascribed to the presence of this compound, was detected in cultures of the ascomycete Botryosphaeria sp. (2) and in basidiomycetes such as Trametes versicolor (39), Nematoloma frowardii (22), Trametes sp. strain I-62 (30), Clitocybula dusenii, and the unclassified strain I63-2 (40). However, other authors have not found any induction of laccase activity after adding this compound to cultures of species such as the basidiomycete PM1 (8), Lentinula edodes (48), and Pleurotus ostreatus (36). Mansur et al. (30) reported increases in both extracellular laccase levels and fungal biomass when Trametes sp. strain I-62 was grown in Kirk medium with 3,4-DMBA. In the present study we confirmed these observations, suggesting that this increase in biomass could be explained if 3,4-DMBA is used as an alternative carbon source.

The chemical structure of an inducer is one of the essential elements determining the inductive capacity of different aromatic compounds on laccase activity (41). The increase in laccase activity could be, among others, the result of transcriptional factors such as an increased production of mRNAs or posttranscriptional factors such as an increased stability of laccase mRNA transcripts. Other options would be a translational control mechanism sensitive to different aromatic compounds or, alternatively, a direct effect of the aromatic compounds on the activity of the enzymes, for example, by increasing their half-life (28). Linden et al. (27) have demonstrated that the induction of laccase in Neurospora crassa is associated with an increase in gene transcription but also involves an mRNA stabilization mechanism, as well as a translational control. From a comparison of 3,4-DMBA with 2,5-DMBA and 3,5-DMBA isomers, it is interesting that compounds which have an identical chemical composition and which differ only in the positions of the functional groups in the aromatic ring can have such profoundly different effects on the enzymatic activity and growth of Trametes sp. strain I-62. This could be explained, perhaps, by the existence of a possible relationship between the efficiency of a compound as a laccase substrate and its capacity to induce the enzyme. Steric differences between isomers can produce changes in their reactivity, which can be a determinant in enzymatic reactions. For instance, groups in the ortho or para position with respect to the hydroxyl groups of phenolic compounds, hydroxindoles, and aromatic compounds favor their oxidation by laccases. However, the presence of various groups, depending on their position and size, may produce the opposite effect due to steric inhibition of the enzyme (6, 12, 29). Specific receptors for phenolic compounds on the fungal hyphae surface of Heterobasidion annosum have been reported (19). If we assume that similar receptors may be present in Trametes sp. strain I-62 to recognize aromatic compounds, the spatial conformation of molecules would be an essential factor in their interaction with the cell.

The inductive effect of 3,4-DMBA, 2,5-DMBA, and 3,5-DMBA was revealed at the level of Trametes sp. strain I-62 lcc gene expression as well. Differential expression of laccase genes has been reported for a few fungi (31, 34, 36, 44, 47, 48). One of these studies, developed in our laboratory, described the differential expression of Trametes sp. strain I-62 lcc1, lcc2, and lcc3 genes in cultures with different carbon source and nitrogen levels (31). lcc3 gene induction by 3,4-DMBA was not detected by Northern blot analysis under different conditions. Here, due to the greater sensitivity of the multiplex RT-PCR approach, we could detect lcc3 and, furthermore, quantitatively compare its expression with that of lcc1 and lcc2 by applying the multiplex RT-PCR technique.

3,4-DMBA, 2,5-DMBA, and 3,5-DMBA produced different induction patterns on the expression of the three laccase genes from Trametes sp. strain I-62. The isomer 2,5-DMBA seems to predominantly induce lcc2, while the most remarkable action of 3,5-DMBA is on lcc1, without ignoring its effect on lcc2 and lcc3 transcript levels. Taking together all the information for the lcc transcript levels in the presence of the three isomers (Fig. 4E), we propose the existence of a signaling mechanism not yet described, modulating and regulating each different laccase gene family member to coordinate and balance the total amounts of laccase transcripts being produced by the fungus at a given time.

As a concluding remark about the technique itself, changes in lcc transcripts can be quickly and easily monitored from 10-mg samples of wet mycelium, facilitating the possibilities of studying gene expression through the analysis of a large number of samples from minimal amounts of mycelia. In addition to being a valuable tool to increase our knowledge about laccase regulation using Trametes sp. strain I-62 as a model, the process could be applied, with minor changes, to study gene expression in different fungal gene families.