Characterization of a Pinus pinaster cDNA encoding an auxin up ...

Tree Physiology 22, 231–238 © 2002 Heron Publishing—Victoria, Canada

Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots V. CHARVET-CANDELA,1,2 S. HITCHIN,1–3 M. S. REDDY,2,4 B. COURNOYER,2 R. MARMEISSE 2 and G. GAY2,5 1

These authors contributed equally to this work

2

Université Claude-Bernard LYON 1, UMR CNRS 5557, Laboratoire d’Ecologie Microbienne, Bât. André Lwoff, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France

3

Present address: School of Natural and Environmental Sciences, Coventry University, Priory Street, Coventry CV1 5FB, U.K.

4

Present address: Thapar Institute of Engineering and Technology, School of Biotechnology, Patiala 147004, India

5

Author to whom correspondence should be addressed ([email protected])

Received March 27, 2001; accepted August 9, 2001; published online February 1, 2002

Summary As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant–microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxinregulated peroxidase during ectomycorrhiza formation. Keywords: differential screening, gene expression, mycorrhiza.

Introduction In temperate forest ecosystems, tree root systems establish symbiotic associations with ectomycorrhizal fungi. This association enhances the growth and fitness of trees by improving

their mineral nutrition and resistance to root pathogens (see review by Smith and Read 1997) and also allows the fungus to complete its life cycle. The differentiation of mycorrhizas follows precise ontogenetic processes leading to integrated symbiotic structures. The development of such integrated structures implies exchange of signalling molecules that coordinate the symbiosis-related alteration of both partners. The role of auxin released by ectomycorrhizal fungi as a signal molecule that regulates root morphogenesis has been suspected since the early work by Slankis (1973), who hypothesized that fungal auxin is responsible, at least in part, for the typical morphology of pine mycorrhizas. This hypothesis was critically assessed by Durand et al. (1992) based on a study of auxin-overproducing mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi. These mutants have increased mycorrhizal activity (Gay et al. 1994), suggesting that indole-3-acetic acid (IAA) is a mediator of fungal infectivity. They also form a hypertrophic Hartig net, and fungal hyphae are able to develop inside living plant cortical cells (Gea et al. 1994), indicating that fungal auxin induces modifications in the host cortical cell wall that favor Hartig net formation (Gay et al. 1995). Although the results obtained with auxin-overproducing mutants strongly suggest the involvement of fungal auxin in the differentiation of symbiotic structures, they do not provide information about the mode of action of fungal auxin. We hypothesized that fungal IAA affects host plant gene expression leading to the developmental modifications observed in IAAoverproducing mutants. To test this hypothesis, we constructed a cDNA library with mRNA extracted from Pinus pinaster (Ait.) Sol. roots treated with auxin (in the absence of fungus). Differential screening of this library led to the identification of three cDNAs up-regulated by auxin, including one cDNA showing homologies with peroxidases. The kinetics, specificity and mechanism of its induction are described.

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fixed on the membranes under UV illumination and each membrane was hybridized to one of the two cDNA probes.

Materials and methods Plant culture and induction Half-sib seeds of P. pinaster, collected in Les Landes, France, were surface sterilized and germinated according to Debaud and Gay (1987). Two-week-old seedlings were transferred to petri dishes containing 45 ml of modified Melin-Norkrans agar medium (Gea et al. 1994) for 2 days at 22 ± 1 °C in a 16-h photoperiod (80 µmol m –2 s –1). Induction treatments were then carried out by soaking the roots of the seedlings in modified Melin-Norkrans liquid medium containing IAA, 2,4-dichlorophenoxyacetic acid (2,4-D), abscisic acid (ABA) or cycloheximide (CHX) at 50 µM concentration, quercetin at 10 µM or ethephon (2-chloroethylphosphonic acid) at 100 µM concentration (for ethylene induction) in the dark at 22 ± 1 °C for 6 h. At the end of the induction treatments, roots were collected and immediately frozen in liquid nitrogen. Construction of cDNA library Total RNA was isolated from roots treated for 18 h with 50 µM IAA as described by Schneiderbauer et al. (1991). A cDNA library in the phage vector λZAP was constructed according to the manufacturer’s instructions (Stratagene, Amsterdam, The Netherlands) from poly(A)+ mRNA purified by two cycles of oligo(dT)-cellulose chromatography (Gibco-BRL, Cergy Pontoise, France). Recombinant λ clones were selected on XGal-isopropylthiogalactosyl NZY agar plates. Mass in vivo excision of the pBluescript plasmid from the Uni-ZAP XR Vector was performed according to the manufacturer’s instructions (Stratagene). Recombinant bacterial colonies containing the pBluescript double-stranded plasmid with the cloned cDNA insert were selected on LB-ampicillin agar plates (50 µg ml –1). Colony and Southern blot differential screening Auxin-induced genes were searched for by differential screening with 32P-labeled cDNA probes prepared from mRNA extracted from (i) auxin-treated and (ii) non-treated roots. The first screening was performed on bacterial colonies. Colonies were incubated overnight at 37 °C on circular 82-mm Nylon membranes (Biodyne A, Pall, Merck Eurolab, Strasbourg, France) placed on LB-ampicillin agar plates (90 cfu/plate). The membranes were placed for 5 min in denaturation solution (0.5 M NaOH, 1.5 M NaCl), 5 min in neutralizing solution (0.5 M Tris-HCl pH 7.4, 1.5 M NaCl) and washed in 2 × SSC (0.3 M NaCl, 30 mM Na citrate pH 7.0). The DNA was fixed on the membranes by incubating at 80 °C for 1 h. Each membrane was successively hybridized to the two cDNA probes. Southern blotting was used to confirm hybridization of colonies only to the probe prepared from RNA extracted from auxin-treated roots. Plasmid DNA was extracted from each of the clones (Sambrook et al. 1989) and digested with EcoRI and XhoI. After electrophoresis in 0.8% agarose gel in 1× TBE (90 mM Tris pH 8, 90 mM boric acid, 2 mM EDTA), gels were blotted in duplicate onto nylon membranes (Hybond-N +, Amersham, Saclay, France) with 0.4 M NaOH. The DNA was

Screening hybridization conditions Membranes were hybridized with [α32P]dCTP-labeled double-stranded cDNA probes (2.0 × 10 8 cpm µg –1) made from total RNA isolated from untreated roots or roots treated with IAA for 18 h. One µg of total RNA was reverse-transcribed and PCR-amplified using the CapFinder PCR cDNA synthesis kit (Clontech, Basingstoke, U.K.), and 250 ng of doublestranded cDNA was radiolabeled with [α32P]dCTP using the Random Priming kit from Boehringer (Mannheim, Germany). Hybridization was performed in 50 mM Tris HCl pH 7.5, 10 mM Na2EDTA, 1 M NaCl, 0.5% SDS, 0.1% Na pyrophosphate (NaPPi), 10 × Denhardt’s solution and 10 mg ml –1 single-stranded DNA at 60 °C. After 48 h, filters were washed twice in 2 × SSC, 0.1% NaPPi, 0.5% SDS for 30 min at 60 °C and once with 0.2 × SSC, 0.1% NaPPi, 0.5% SDS for 30 min at 60 °C before exposure to X-OMAT Kodak films between intensifying screens at –70 °C. Sequence analyses Plasmid DNA was isolated on QIAGEN columns (Plasmid Midi kit) and the insert was sequenced on both strands (Genome Express, Grenoble, France) with the T3 and T7 primers. The DNA homology searches were performed against protein and nucleotide databases using the BLAST2 program (Altschul et al. 1997) through the National Center for Biotechnology Information. Protein sequences were aligned with the multiple alignment CLUSTALW software (Higgins and Sharp 1988). Phylogenetic analyses were performed with the PHYLO-WIN package (Galtier et al. 1996). The neighbor-joining method was used to build a phylogenetic tree from the Poisson or observed divergence percentage distance matrices (Saitou and Nei 1987). The significance of internal branches of the phylogenetic tree was assessed by applying the observed divergence method to 1000 bootstrap replicates (Felsenstein 1985). The PpPrx75 sequence appears in the DDJB/GenBank EMBL databases under Accession Number AJ251254. RT-PCR analysis of PpPrx75 expression Initial attempts to quantify PpPrx75 gene expression by Northern blot analysis gave faint hybridization signals that could not be quantified. To circumvent this problem we performed mRNA quantifications by RT-PCR. Total RNA was extracted by the method of Keifer et al. (1999), which includes a DNase I treatment. Reverse transcription was performed in a final volume of 25 µl at 42 °C for 1 h with 2 µg total RNA, 10 mM of each dNTP, 25 U of RNase inhibitor (RNasin, Promega, Lyon, France), 0.5 µg oligo(dT)15, 200 U of M-MLV reverse transcriptase (Promega) and the appropriate buffer. Absence of contaminating genomic DNA was confirmed on each sample by amplifying a control gene with primers flanking an intron sequence (data not shown). The PCR reactions were performed in a final volume of 50 µl using 2 µl of a quarter-strength cDNA sample, 2 mM MgCl2, 100 µM dNTPs,

TREE PHYSIOLOGY VOLUME 22, 2002

CLONING OF A PEROXIDASE cDNA IN PINUS PINASTER

200 nM of each primer, 2 U of Taq polymerase and the appropriate buffer (Gibco-BRL). Amplification conditions consisted of 3 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C followed by 5 min at 72 °C. Two distinct cDNA fragments were amplified simultaneously: a 651-bp fragment from the PpPrx75 gene (using primers 5′-GT CGAAACAAAATGCCCG-3′ and 5′-TCAATTCACGGCTC TGCA-3′) and a 357-bp fragment from the Pp139 gene (primers 5′-TTAAGGATCGCCTTCTTC-3′ and 5′-GGCAATCCT TGCTAAAGTATTC-3′). The expression of this latter gene is not affected by auxin treatment and served as an internal control for RNA extraction, reverse transcription and PCR reactions. The PCR fragments were separated on 2% agarose gels and the ethidium bromide intensity of each band quantified with Molecular Analyst software (Bio-Rad, Hercules, CA). A negative control without added cDNA was included in all PCR experiments. Because these controls were always negative, they are not presented in the figures. Plant induction was performed in duplicate or triplicate based on 10 seedlings per RNA extraction. The cDNA was synthesized once or twice from each RNA extraction followed by two to four PCR amplifications for each cDNA sample.

Results Construction and differential screening of a cDNA library A cDNA library in the expression vector λZAP was prepared from poly(A)+ mRNA isolated from P. pinaster roots treated for 18 h with 50 µM IAA. About 8.3 × 10 5 recombinant clones were obtained. Analysis of 200 phage inserts amplified by PCR using T3 and T7 showed that the mean insert size was 1.4 kb (minimum 0.15 kb, maximum 4.3 kb). Mass excision of pBluescript phagemids was performed and a total of 1368 colonies were recovered for further analyses. These colonies were screened against labeled cDNAs derived from either auxin-treated or untreated roots. Secondary differential screening of clones showing stronger hybridization signals with the auxin-treated root cDNAs than with the untreated root cDNAs led to the identification of three IAA up-regulated cDNAs: D75, C61 and C16 (Figure 1). Identification of a P. pinaster peroxidase encoding cDNA One cDNA insert, PpPrx75 (D75 in Figure 1), was fully sequenced on both strands. This cDNA is 788 nucleotides long, excluding the poly(A)+ tail (Figure 2), and contains a single open reading frame encoding 216 amino acids. A BLASTP search with the deduced amino acid sequence revealed homology to more than 50 plant peroxidases. Sequence identities of the PpPrx75 clone with these peroxidases were above 42%. The PpPrx75 cDNA is related to four cationic peroxidase cDNAs isolated from Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK, sharing between 46 and 52% amino acid sequence identity and 63 to 68% similarity (Figure 3). Although the overall amino acid sequence identities of PpPrx75 to other peroxidase genes were less than 52%, there

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Figure 1. Identification of auxin-induced cDNA library clones. After two rounds of differential screening, three positive cDNA clones were identified: D75, C61 and C16. Southern blot analyses of these EcoRI and XhoI pBluescript inserts are presented: (a) hybridized with cDNA from auxin-treated roots, and (b) hybridized with cDNA from untreated roots. Arrowheads indicate hybridizations with inserts and the asterisk indicates aspecific hybridizations to the pBluescript plasmid.

was a high degree of identity in the conserved domains that are characteristic of plant peroxidases. The first domain (Residues 56–77 in A. thaliana 1) could not be found in PpPrx75, suggesting that the cDNA is not full length. In the second domain (Residues 5–28 in P. pinaster), P. pinaster cDNA shares 79% identities with Arabidopsis and Arachis peroxidases; the identity with Stylosanthes is 75%. Noteworthy is the evident conservation of the VSCADI sequence in the second domain. In the third domain (Residues 34–40), the sequences are fully conserved. The fourth domain (Residues 75–84) shows the greatest variability, with identities ranging from 60 to 90%. The amino acid sequence predicted from the pine cDNA clone possesses five cysteine residues indicated by closed circles in Figure 3. Their positions correspond well with the disulfide bridge-forming cysteines found in peroxidases. One histidine residue (indicated by an open circle in Figure 3) involved in heme binding (Buffard et al. 1990) was also identified. The PpPrx75 contains a possible glycosylation site (NTS, Residues 138–140) that is not found in other peroxidases. The sequences between conserved domains are similar in length across the different peroxidases examined. The sequence homology of PpPrx75 with cationic peroxidases together with the alkaline pI (8.5) of the deduced polypeptide sequence supports the idea that this gene encodes a cationic isozyme. Phylogenetic analysis The PpPrx75 deduced amino acid sequence was compared with those of 19 cationic peroxidases cloned from a wide range of angiosperms belonging to different families of either monocots or dicots. The multiple alignment derived from these sequences contained 168 variable sites including 152 informative ones. Neighbor-joining phylogenetic trees derived from distances computed by the Poisson method or according to the observed percentage of divergence have identical topol-

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Figure 2. Nucleotide and deduced amino acid sequences of the Pinus pinaster PpPrx75 cDNA. Underlined nucleotides indicate the location of primers used for RT-PCR.

ogies. The observed divergence tree is shown in Figure 4. The PpPrx75 belongs to a cluster containing two A. thaliana cationic peroxidases that have likely emerged from a duplication. Several distinct and significant phylogenetic lineages were observed among this group of peroxidases. The phylogenetic tree could be divided into at least six distinct clusters, each cluster having likely emerged after ancestral duplication events. For example, only a duplication event could have led to Clusters II and III because they both contain a sequence found among Nicotiana tabacum L. This is also true for the divergence of Clusters V and VI, which both contain sequences from Papilionoideae. Differential expression of PpPrx75 mRNA in pine roots Differential accumulation of PpPrx75 mRNA was detected 3 h after induction with 50 µM IAA (Figure 5). Transcript accumulation increased for the next 24 h. On average, the amount of transcript was about 1.5 times higher in induced roots than in untreated roots. The PpPrx75 mRNA accumulated in P. pinaster roots following a 6-h treatment with active auxins including IAA and 2,4-D (Figure 6) or with other hormones such as ethylene or ABA. Quercetin, a flavonoid possibly involved in plant–microorganism interactions, also induced PpPrx75 transcript accumulation but to a lesser extent. In an attempt to clarify the mode of regulation of PpPrx75, we studied the effect of the protein synthesis inhibitor cycloheximide (CHX) on mRNA accumulation. Transcript differ-

ential accumulation was identical in roots treated either with IAA alone or with IAA + CHX (Figure 7), indicating that IAA induction of PpPrx75 did not require protein synthesis because CHX did not inhibit IAA-induced mRNA accumulation. Cycloheximide alone also induced mRNA accumulation but to a lesser extent than IAA.

Discussion This study was undertaken to identify host plant genes up-regulated by fungal auxin during ectomycorrhiza formation. A cDNA library was constructed from auxin-treated P. pinaster roots. Differential screening of this library allowed the identification of a cDNA encoding a putative cationic peroxidase. An amino acid sequence alignment including this novel sequence and cationic plant peroxidases showed that the domains essential for enzyme activity are present and highly conserved in the pine polypeptide that is closely related to Arabidopsis cationic peroxidases. A phylogenetic gene tree derived from this multiple alignment showed that this group is characterized by several significant clusters with rather long terminal branches. Polypeptides from the same organism (either A. thaliana or N. tabacum) or from species belonging to a subfamily (A. hypogaea, Glycine max (L.) Merrill, Medicago sativa L., S. humilis and Trifolium repens L. belonging to the Papilionoideae) are not grouped in the same clades. This could be explained by a major ancestral phylogenetic burst that would have given rise to several alleles of the cationic peroxi-



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Figure 3. Alignment of the deduced amino acid sequence of the Pinus pinaster PpPrx75 cDNA with those of other cationic plant peroxidases. Gaps introduced to produce the best alignment are indicated by dashes. Identical amino acid residues are boxed. Gray boxes are functionally similar amino acid residues. Conserved cysteine residues forming disulfide bridges and histidine residues essential for catalysis are indicated by closed and open circles, respectively. A potential glycosylation site (N-X-S/T, where X is any amino acid) is indicated by an asterisk. Abbreviations: At1 = peroxidase cDNA clone from Arabidopsis thaliana, Accession No. X98322 (Capelli et al. 1996); At2 = putative peroxidase from Arabidopsis thaliana, Accession No. AL049483 (M. Bevan et al., Max Planck Institut für Biochemie, Martinsried, Germany, unpublished data); Ah = cationic peroxidase from Arachis hypogaea, Clone PNC2, Accession No. M37637 (Buffard et al. 1990); and Sh = pathogen-inducible cationic peroxidase gene from Stylosanthes humilis, Accession No. L77080 (Stines et al. 1996).

dase genes early in the evolution of green plants. Each duplicate copy would have evolved independently from the others, with some being deleted over time, thus creating distinct gene families whose members are distributed across the whole plant kingdom. The cloned pine peroxidase would therefore belong to a subfamily that comprised a peroxidase gene from A. thaliana that would have been duplicated later on. In pine roots, the PpPrx75 peroxidase gene is up-regulated following exogenous IAA supply, in contrast with studies showing that total peroxidase activity is generally down-regulated by auxins (Klotz and Lagrimini 1996). These results are not contradictory because electrophoretic studies of Catharanthus peroxidases showed that IAA differentially affected the different isoforms, some of them being down-regulated,

others being up-regulated (Morgens et al. 1990, Limam et al. 1997). Considering that some peroxidase isozymes can also be induced by ethylene (Ishige et al. 1993), the up-regulation of PpPrx75 in auxin-treated P. pinaster roots might be due to either a direct effect of auxin or ethylene synthesized in roots in response to IAA treatment. To clarify this point, we studied the specificity of PpPrx75 induction. It appeared that it was not specifically induced by IAA. In particular, it could be induced by ethylene. For this reason, the involvement of ethylene as a second messenger in IAA-induced PpPrx75 mRNA accumulation in pine roots could not be ruled out. This question was further examined using the protein synthesis inhibitor cycloheximide. Cycloheximide did not inhibit IAA-induced transcript accumulation, indicating that PpPrx75 induction by


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Figure 4. Neighbor-joining phylogenetic tree of cationic plant peroxidases. One hundred and sixty eight variable sites, of which 152 are cladistically informative, were used. Distances are proportional to evolutionary divergences expressed in substitution per 100 sites and are indicated on the tree branches. Bootstrap values are indicated in the ovals. Plant species belonging to the Papilionoideae subfamily are indicated by a star. The accession numbers are provided next to plant names.

IAA does not require protein synthesis. Therefore, we conclude that it is a primary (direct) response to auxin that is independent of ethylene biosynthesis. Cycloheximide alone also induced PpPrx75 mRNA accumulation, but to a lesser extent than IAA. This induction can be ascribed either to the inhibition of the synthesis of a short-lived inhibitory protein or to the stabilization of mRNAs through the inhibition of RNAse synthesis. Because cycloheximide induction was less than IAA induction, we speculate that it was associated with mRNA stabilization, rather than with inhibition of the synthesis of a repressor protein. Similarly, we postulate that accumulation of PpPrx75 mRNA following auxin treatment was a result of the induction of the corresponding gene, rather than its derepression. Altogether, these results suggest that, in ectomycorrhizas, PpPrx75 could be up-regulated as a primary response to fungal IAA. It might also be regulated by other hormones such as ABA or ethylene or by non-hormonal compounds such as quercetin. Comparison between mycorrhizas

formed by wild-type strains and IAA-overproducing fungal mutants (Gay et al. 1994, Gea et al. 1994) will be used to determine whether PpPrx75 is induced as a primary response to fungal auxin during ectomycorrhiza formation. Concerning the possible role of PpPrx75 in ectomycorrhizas, we note that plant peroxidases are usually encoded by a family of genes and are ubiquitous in the plant kingdom. In general, cells contain a large number of peroxidase isozymes, each with a broad catalytic specificity. Peroxidases play a key role in many different physiological processes such as lignification, suberization, organogenesis, aging, wound healing and auxin metabolism. Furthermore, peroxidases are implicated in plant–microorganism interactions (see Gaspar et al. 1991). Peroxidases are also involved in plant defense mechanisms against pathogens and their activity in plants usually increases following infection. In symbiotic associations, the role of peroxidases is unclear. Peroxidases are involved in Rhizobium nodule formation (Cook et al. 1995, Peng et al. 1996), their



Figure 5. Kinetics of PpPrx75 mRNA accumulation in response to IAA. Pinus pinaster roots were incubated in the presence (■) or absence (▲) of 50 µM IAA. After the indicated period, 2 µg total RNA was used for RT-PCR. Multiplex PCR was performed using two primer combinations. The first was specific for PpPrx75, whereas the second was specific for Pp139, which is constitutively expressed in roots and unaffected by hormonal treatments. The latter was used as an internal standard for PpPrx75 PCR product quantification. Agarose gel photograph shows ethidium bromide-stained RT-PCR products (651 bp for PpPrx75 and 357 bp for Pp139) amplified from cDNA synthesized from total RNA extracted from pine roots. Vertical lines represent standard deviation of the mean (P < 0.05).

activity being generally higher in nodules than in roots (Staehelin et al. 1992). The involvement of peroxidases in the mycorrhizal symbiosis remains largely unknown. Ronald and

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Figure 7. Effect of the protein synthesis inhibitor cycloheximide (CHX) on the induction of PpPrx75. Pinus pinaster roots were treated for 6 h with either 50 µM indole-3-acetic acid (IAA), 50 µM IAA + 50 µM CHX (IAA + CHX) or 50 µM CHX alone (CHX). Control (C) consisted of untreated roots. See Figure 5 for quantification approach and standard deviation.

Söderhäll (1985), Spanu and Bonfante-Fasolo (1988) and Timonen and Sen (1998) did not detect significant variation in total peroxidase activity in roots following mycorrhizal association. However, a decrease in peroxidase activity in mycorrhizal roots has been reported by Bonfante-Fasolo and Scannerini (1980) and by Tarkka et al. (2001), whereas Albrecht et al. (1994) and Mathur and Vyas (1995) reported an overall increase in peroxidase activity in mycorrhizal root systems. Isozyme studies by Spanu and Bonfante-Fasolo (1988), Mathur and Vyas (1995) and Timonen and Sen (1998) showed that these contradictory results may be ascribed to the presence of multiple peroxidase isoforms that can be regulated independently. We conclude that to understand the role of peroxidases in the mycorrhizal symbiosis, each peroxidase isozyme should be considered separately. The cDNA clone characterized in the present study is a suitable tool for studying the expression of a peroxidase isozyme during mycorrhiza differentiation. Acknowledgments The authors are grateful to C. Raffier for her valuable technical assistance. A part of this work was supported by the Indo-French IFCPAR program. V. Charvet-Candela was supported by a grant from the Rhône-Alpes Region and by a short-term mission from the EC COST action E6 Eurosilva.

Figure 6. Specificity of the induction of PpPrx75 mRNA accumulation. Pinus pinaster roots were incubated for 6 h with indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) or abscisic acid (ABA) at 50 µM concentration, 100 µM ethephon (Eth) or 10 µM quercetin (Que). Untreated roots served as a control (C). See Figure 5 for quantification approach and standard deviation.

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