Mycorrhiza

, Volume 17, Issue 4, pp 327–335 | Cite as

GintMT1 encodes a functional metallothionein in Glomus intraradices that responds to oxidative stress

  • M. González-Guerrero
  • C. Cano
  • C. Azcón-Aguilar
  • N. Ferrol
Original Paper

Abstract

A full-length metallothionein (MT) gene (GintMT1) was isolated from Glomus intraradices extraradical mycelium. This is the first MT gene reported in the genus Glomus, third in the Glomeromycota. Functional analysis of GintMT1 in a MT-defective Saccharomyces cerevisiae strain indicates that it encodes a functional MT. Gene expression analyses revealed that the transcript levels of GintMT1 were elevated in mycelia treated with 5 mM Cu or paraquat but inhibited in mycelia treated with 50 μM Cu or 450 μM Cd. The elevated expression of GintMT1 in the 5 mM Cu-treated mycelia together with the ability of GintMT1 to provide tolerance to a Cu-sensitive yeast suggests that GintMT1 might afford protection against Cu. Induction of GintMT1 expression by paraquat and 5 mM Cu, treatments that also produced an oxidative damage to the fungal membranes, suggests that GintMT1 may play a role in the regulation of the redox status of the extraradical mycelium of G. intraradices.

Keywords

Arbuscular mycorrhizal fungi Heavy metals Lipid peroxidation Metallothionein Oxidative stress 

Introduction

Various heavy metals (HM), such as Zn, Cu, Mn, Ni, and Co, are essential micronutrients required for development of living organisms, whereas others such as Cd, Pb, and Hg, have no known biological function and are toxic even at very low concentrations. Accumulation of HM in the soil from either natural sources such as volcanoes and continental dusts, or human activities like mining, combustion of fossil fuels, metal-working industries, etc., have detrimental effects on soil organisms and therefore, on ecosystem functioning.

There is evidence that arbuscular mycorrhizal (AM) fungi are present in HM polluted sites (Weissenhorn et al. 1995; del Val et al. 1999a; Chen et al. 2003), and several studies have shown a positive impact of mycorrhizal establishment on plant resistance to HM, the resulting effect depending on plant growth conditions, AM fungal isolate involved, and HM concentration (del Val et al. 1999b; Hildebrandt et al. 1999; Rivera-Becerril et al. 2002). The occurrence of AM fungi in metal-contaminated soils provides evidence of adaptation and tolerance of these microorganisms to toxic metals, although it has been shown that this naturally occurring tolerance decreases after a few generations in the absence of HM (Malcová et al. 2003). The constitutive and adaptive mechanisms of mycorrhizas to contaminated soils and the contribution of AM symbiosis to soil remediation have been recently reviewed (Meharg 2003; Gohre and Paszkowski 2006). Nevertheless, current knowledge on the molecular basis of HM tolerance in AM fungi is still in its infancy.

AM fungi, like other organisms, must have evolved a range of mechanisms including binding to extracellular materials, regulation of transport processes, and/or intracellular sequestration to maintain the concentration of essential metals within a physiological range and control the toxicity of nonessential HM ions. The ability of the AM fungal hyphae to sequester HM not only in the mucilaginous zone of the hyphal wall and in the cell wall but also inside the hyphal cytoplasm has been described (González-Chavez et al. 2002). The finding that glomalin, a glycoprotein copiously produced by AM fungi, efficiently sequesters both in vivo and in vitro different HM has led to hypothesize that glomalin may be one of the molecules involved in HM sequestration both at the hyphal cell wall and in the soil (González-Chavez et al. 2002). At the genetic level, just two genes involved in HM homeostasis have been analyzed in detail in AM fungi: the Zn transporter GintZnT1 from Glomus intraradices involved in vacuolar Zn compartmentalization (González-Guerrero et al. 2005) and the metallothionein gene GmarMT1 from Gigaspora margarita that may provide protection against Cu (Lanfranco et al. 2002). Recently, several as-yet-uncharacterized gene sequences coding for enzymes potentially catalyzing the detoxification of reactive oxygen species were found in a suppression subtractive hybridization library from hyphae of G. intraradices grown in high versus low Zn (Ouziad et al. 2005).

Metallothioneins (MTs) constitute an extensive and diverse family of small cysteine-rich proteins that bind metals via the thiol groups of their cysteine residues and may play a role in the intracellular sequestration of HM. MTs are distributed throughout all the kingdoms, and have been traditionally classified, based on the arrangement of their cysteine residues, into two classes (Fowler et al. 1987; Kojima 1991). Class I MTs are widespread in vertebrates, whereas class II MTs include all those from plants and fungi as well as from nonvertebrate animals. Because MTs efficiently bind metals and some MT genes are positively regulated by metals, they are thought to play a role in cellular HM detoxification and homeostasis (Cobbett and Goldsbrough 2002). Nonetheless, a growing amount of evidence suggests an alternative role for MTs as protectants from oxidative damage (Tamai et al. 1993), a function that results from the capability of the thiolate groups to be reversible oxidized (Maret 2003). With the aim of getting further insights into the putative roles of MTs in AM fungi, regulation of a new MT gene from G. intraradices by Cu and Cd was analyzed, and the possibility that its induction could be linked to oxidative stress caused by these metals was studied.

Materials and methods

AM monoxenic cultures

AM monoxenic cultures were established as described by St-Arnaud et al. (1996). Briefly, a carrot (Daucus carota L., clone DC2) Ri-T DNA transformed root organ culture was grown together with the AM fungus G. intraradices Smith & Schenck (DAOM 197198, Biosystematic Research Centre, Ottawa, Canada) in two-compartment Petri dishes. Cultures were initiated in the “root compartment”, which contained a minimal culture medium (“M medium”) gelled with 0.35% (w/v) phytagel (Sigma-Aldrich, St. Louis; Chabot et al. 1992). Fungal hyphae, but not roots, were allowed to grow over the plastic barrier to the second compartment (the “hyphal compartment”), which contained the gelled M medium described above but without sucrose (“M-C medium”; St-Arnaud et al. 1996). Plates were incubated in the dark at 24°C.

Treatments of G. intraradices extraradical mycelium in monoxenic cultures

Cu, Cd, or paraquat, an oxidative agent, were applied as a pulse to the M-C medium once the extraradical mycelium was well established on the hyphal compartment (transition from absorptive to sporulative phase, typically after 7–8 weeks after culture establishment). CuSO4, CdSO4, or paraquat were applied to their respective hyphal compartments to obtain final concentrations of 50 μM Cu, 5 mM Cu, 0.45 mM Cd, or 500 μM paraquat. This was done by distributing 500 μl of each filter-sterilized stock solution drop wise so that even diffusion of the added solutions was ensured. Time point just before HM/paraquat addition is referred to “Time 0.” After the pulse, the extraradical mycelium was harvested at time points 12 h, 24 h, and 7 d. Control plates received 500 μl sterile H2O.

Extraradical mycelia recovery and RNA extraction

Extraradical mycelium from the different hyphal compartments was recovered by blending (5 sec. high speed, 5 min. with occasional low speed pulses) the culture medium in 10 mM sodium citrate (pH 6) and collecting the mycelia with a 50 μm sieve under sterile conditions (Bago et al. 1999). The mycelium was immediately liquid nitrogen frozen and stored at −80°C until used. RNAs were extracted using the RNeasy Plant Mini Kit (QIAGEN, Maryland, USA) following manufacturer’s instructions.

Gene isolation, cloning, and sequence analyses

The 3′ end of GintMT1 was identified in the G. intraradices EST database (Accession number BI452270) and obtained from Dr. M. Harrison (Noble Foundation, Ardmore, OK, USA). The 5′ end was obtained by rapid amplification of cDNA ends (RACE) using the SMART RACE cDNA Amplification kit (Clontech, Palo Alto, CA, USA), the GintMT1-specific primer MT1 5′-AGTTACATGCTCCTGGAGAACACTT-3′ and 1 μg total RNA from extraradical mycelia grown in control plates. The amplified cDNA was cloned in the pGEM-T Easy vector (Promega, Madison, USA). GintMT1 was then excised from the pGEM-T Easy vector by NotI digestion and cloned into the NotI-linearized yeast expression vector pFL61 (Minet et al. 1992).

Nucleotide sequences were determined by Taq polymerase cycle sequencing by using an automated DNA sequencer (Perkin-Elmer ABI Prism 373). Computer database comparisons were performed using Basic Local Alignment Search Tool (BLAST) algorithm (Altschul et al. 1990) and computer translation by using the translate tool from EXPASY Molecular Biology Server. Amino acid sequence comparisons were made with the BESTFIT program of the Genetics Computer Group (Madison, WI, USA). Multiple sequence alignments of translated gene sequences were carried out with the program CLUSTALW (version 1.5; Thompson et al. 1994).

Functional complementation assays in yeast

The ΔCUP1 strain of Saccharomyces cerevisiae DTY113 (α ura3-50 leu2-3,112 trp1-1 gal1 his Δcup61; Ecker et al. 1986) was transformed with pFL61 (negative control) or a properly oriented pFL61-GintMT1 construct. Transformants were selected by auxotrophy to uracile in SD medium. For the metal tolerance tests on agar plates, a drop (105 cells) of a liquid culture of the transformants in selective media was placed on SD plates containing or not containing 50 μM CdSO4 or 500 μM CuSO4. Plates were grown aerobically for 3 d at 30°C.

Gene expression analyses

GintMT1 gene expression was studied by real-time polymerase chain reaction (RT-PCR; iCycler iQ, BioRad, Hercules, CA, USA). cDNAs were obtained from 1 μg of DNase-treated total RNA from the different treatments in a 20 μl reaction containing 200 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 100 ng of random hexamers, according to the manufacturer’s protocol. The primer set used to amplify GintMT1 in the synthesized cDNAs were MT1 and MT2 (5′-GGAGATAGTTGTCAATGTACAGGTG-3′). Each 25-μl reaction contained 1 μl of a 1:10 dilution of the synthesized cDNA, 200 mM dNTPs, 200 nM each primer, 3 mM MgCl2, 2.5 μl 1 × SyBR Green (Molecular Probes, Eugene, OR, USA), and 0.5 U Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) in 1 × PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl).

The PCR program consisted of 5 min incubation at 95°C to activate the hot-start recombinant Taq DNA polymerase, followed by 35 cycles of 30 sec at 95°C, 45 sec at 55°C and 45 sec at 70°C, where the fluorescence signal was measured. The specificity of the PCR amplification procedure was checked with a heat dissociation protocol (from 70 to 100°C) after the final cycle of the PCR. The efficiency of the primer set was evaluated by performing RT-PCR on several dilutions of plasmid DNA. The results obtained for the different treatments were standardized to the 18S rRNA levels, which were amplified with the primers: RMF: 5′-TGTTAATAAAAATCGGTGCGTTGC-3′ and RMR: 5′-AAAACGCAAATGATCAACCGGAC-3′. RT-PCR determinations were performed on three independent biological samples from three replicate experiments. Three technical replicates for each biological sample were included in the iCycler. The relative levels of transcription were calculated by using the \(2^{{ - \Delta \Delta {\text{Ct}}}} \) method (Livak and Schmittgen 2001). In all RT-PCR reactions, a non-RT control was used to detect any possible DNA contamination.

Lipid peroxidation

Lipid peroxidation was measured by the thiobarbituric acid-reacting substances method (Beuge and Aust 1978). Briefly, 20 mg extraradical mycelium were homogenized in 60 μl of sample buffer containing 50 mM potassium phosphate pH 7.8, 10% glycerol, 0.1 mM ethylenediamine tetraacetic acid, 0.1% Tritón X-100, and 5 mM Dithiothreitol. The suspension was filtered through two layers of Miracloth and centrifuged for 20 min at 27,000 g at 4°C. The supernatant was collected, added to five volumes of a solution consisting of 15% trichloroacetic acid, 0.375% thiobarbituric acid, and 0.01% hydroxytoluene butilate in 0.25 N HCl, and incubated for 15 min in boiling water. Samples were then left to cool and centrifuged for 5 min at 2,500 g. The amount of malondialdehyde reacting with thiobarbituric acid in the supernatant was determined by measuring the OD at 535 nm. Protein content was determined by the method of Bradford (1976) using BSA to standardize the assay. The amount of protein was used to normalize the peroxidation values. Data are means of three independent experiments carried out on three different biological samples.

Results

Sequence analysis of GintMT1

A 335 bp cDNA fragment corresponding to the 3′ end of a putative MT gene was identified in a G. intraradices EST database (accession number BI452270) and obtained from Dr. M. Harrison (Noble Foundation, Ardmore, OK, USA). The 5′end of this gene was obtained by 5′-RACE using a gene-specific primer and cDNA from G. intraradices extraradical mycelium as template. The full-length gene, named GintMT1, contained a 213-nucleotide open reading frame, a 5′UTR of 73 bp, and a 3′UTR of 187 bp. Several stop codons upstream the ATG initiation codon and the presence of a TGA stop codon well before the end of the cDNA indicates that the cDNA sequence is indeed the full length (Fig. 1a). The deduced amino acid sequence of GintMT1 consists of 71 amino acids with a predicted molecular mass of 7.2 kDa, and containing 13 cysteine residues (Fig. 1a). BLAST searches in the protein sequence database indicated that GintMT1 is related to gene members of the MT family, showing 55 and 48% similarity to the MT genes of the AM fungi Gigaspora rosea and G. margarita (Stommel et al. 2001; Lanfranco et al. 2002), respectively.
Fig. 1

a Nucleotide and deduced amino acid sequence of GintMT1. The two cysteine-rich domains appear underlined. b Multiple alignment of GintMT1 with other known fungal MT genes (GintMT1 Acc. no. AM040753; GmarMT1 Acc. no. AJ421527; GrosMT1 Acc. no. BE057027, and CcMT Acc. no. DY852723)

GintMT1 contains two cysteine-rich domains separated by a 24-amino acid region. The N-terminal domain contains six cysteine residues showing the following pattern: Cys-X-Cys-X3-Cys-X-Cys-X3-Cys-X-Cys, and the C-terminal domain presents seven cysteine residues arranged as follows: Cys-X4-Cys-X-Cys-X2-Cys-X-Cys-X4-Cys-X-Cys (Fig. 1a). This sequence type fits with the pattern presented by class II MTs. The GintMT1 cysteine arrangement is closely shared by the other two known mycorrhizal MTs and by a putative MT from Coprinus cinereus (Fig. 1b), resulting in the conserved arrangements Cys-X-Cys-X3-Cys-X-Cys-X2–3-Cys-X-Cys at the N terminus and Cys-X4-Cys-X-Cys-X2–3-Cys-X-Cys-X4-Cys-X-Cys at the C terminus. Additionally, some ESTs from ectomycorrhizal fungi encoding putative MTs such as the EST clone AAS19463 from Paxillus involutus or the Hebeloma cylindrosporum sequence CK995302, have the same C-terminal domain (data not shown).

GintMT1 encodes a functional MT

The ability of GintMT1 to function as a MT was assessed in a yeast strain carrying a deletion of the MT gene CUP1, mutation which renders the yeast strain sensitive to both Cu and Cd (Ecker et al. 1986). As shown in Fig. 2, transformation of this yeast strain with a plasmid carrying the GintMT1 gene under the control of the constitutive yeast phosphoglycerate kinase promoter complemented the defect of the ΔCUP1 mutant to grow in media containing 500 μM Cu or 50 μM Cd. Restoration by GintMT1 of the function lost by the CUP1 deletion indicates that GintMT1 encodes a functional MT that is able to sequester metal ions.
Fig. 2

Complementation of a yeast strain defective at the MT locus CUP1 by GintMT1. DTY113 cells were transformed with pFL61-GintMT1 or with the empty vector pFL61 and grown in SD medium supplemented with 50 μM Cd or 500 μM Cu

HM regulation of GintMT1 expression

To investigate whether GintMT1 could play a role in HM detoxification/homeostasis in G. intraradices, GintMT1 expression was analyzed in extraradical mycelium that had been exposed to Cu (50 μM and 5 mM) or Cd (0.45 mM) for 12 h, 24 h, and 7 d. As GintMT1 expression in mycelia from control plates was the same at all the time points analyzed, gene expression data are referred to the expression levels detected in mycelia from control plates at time 0. Exposure of extraradical mycelia to 50 μM Cu inhibited transiently GintMT1 expression (Fig. 3a). Relative to the control mycelia, GintMT1 transcript levels decreased 12 and 24 h after the addition of 50 μM Cu but returned to the levels detected in the untreated mycelia 7 d after metal addition. However, exposure to 5 mM Cu clearly induced an increase in GintMT1 transcripts. The level of these transcripts was already slightly higher 24 h after the addition of 5 mM Cu (1.8-fold increase) and further increased (fivefold) 7 d later. By contrast, exposure of the extraradical mycelium of G. intraradices to Cd inhibited GintMT1 transcription at all time points examined.
Fig. 3

a Time course analysis of GintMT1 expression in G. intraradices extraradical mycelium grown in M-C medium after the addition of 50 μM Cu, 5 mM Cu or 0.45 μM Cd. b Time course analysis of GintMT1 expression in G. intraradices extraradical mycelium grown in M-C medium after the addition of 500 μM paraquat. Gene expression was studied by RT-PCR using specific primers for 18S rRNA and GintMT1. The relative levels of transcription were calculated by using the 2-ΔΔ CT\(2^{{ - \Delta \Delta }} {\text{C}}_{{\text{T}}} \) method. Bars represent SD of the means of three independent experiments

Regulation of GintMT1 by oxidative stress

To gain more information about the putative roles of GintMT1 and taking into consideration that MTs may also play a role in scavenging reactive oxygen species, the effect of paraquat, an intracellular superoxide generator, on GintMT1 expression was analyzed. Addition of paraquat to the extraradical mycelium of G. intraradices clearly induced an increase of GintMT1 transcript levels at all time points analyzed. A twofold induction was detected 12 and 24 h after the addition of paraquat, and a ninefold up-regulation was observed after 7 d exposure (Fig. 3b). These data suggest that GintMT1 may act as an antioxidant in G. intraradices.

Analysis of the oxidative stress caused by HM exposure

The finding that GintMT1 transcript levels increased in mycelia exposed to paraquat and the consideration that some HM have been reported to cause oxidative stress in different organisms (de Vos et al. 1992; Gallego et al. 1996; Yamamoto et al. 1997) prompted us to check whether regulation of GintMT1 gene expression by Cu and Cd could be linked to the oxidative status of the fungus.

Oxidative stress was estimated by measuring the level of oxidative damage of membrane lipids in the Cu- and Cd-treated mycelia, more precisely by using the levels of malondialdehyde formed by the alteration of polyunsaturated acids due to peroxidation. In control mycelia, lipid peroxidation did not change during the time-course experiment (data not shown). Relative to the control mycelia, addition of 5 mM Cu caused a great increase in lipid peroxidation at all time points analyzed (Fig. 4a); however at 50 μM Cu, lipid peroxidation was only increased 1 week after the addition of the metal. Exposure of G. intraradices extraradical mycelia to Cd increased the level of lipid peroxidation at all time points analyzed, being this increase higher 24 h post-addition of the metal. As expected, exposure of the mycelia to paraquat clearly increased lipid peroxidation (Fig. 4b).
Fig. 4

a Lipid peroxidation levels in G. intraradices extraradical mycelium grown in M-C medium after the addition of 50 μM Cu, 5 mM Cu or 450 μM Cd. b Lipid peroxidation levels in G. intraradices extraradical mycelium grown in M-C medium after the addition of 0.5 mM paraquat. Data are expressed as the relative amount of malondialdehyde referred to the controls. Bars represent SD of the means of three independent experiments

Discussion

The results presented in this paper describe the characterization of a new MT gene from G. intraradices that provides tolerance to a Cu-sensitive yeast strain and is transcriptionally regulated by Cu, Cd, and paraquat in the extraradical mycelia of G. intraradices.

Although the most widely accepted role for MTs is metal detoxification, several studies have indicated that MTs play a role in the protection against the effect of reactive oxygen species. In fact, the MT protein itself acts as an antioxidant as it is a potent scavenger of hydroxyl radicals (Andrews and Geiser 1999). Our expression analyses revealed that the transcript levels of GintMT1 were elevated in mycelia treated with 5 mM Cu or paraquat but inhibited in mycelia treated with 50 μM Cu or 450 μM Cd. The elevated expression of GintMT1 in the 5 mM Cu-treated mycelia together with the ability of GintMT1 to provide tolerance to a Cu-sensitive yeast suggests that GintMT1 might afford protection against Cu, as it was hypothesized for the MT gene GmarMT1 of G. margarita (Lanfranco et al. 2002). However, down-regulation by a lower Cu concentration as well as by Cd suggests that the main role of GintMT1 in G. intraradices is not related to HM detoxification, as it could be concluded from the yeast complementation assays showing the ability of GintMT1 to bind both Cu and Cd. Up-regulation of GintMT1 expression by paraquat indicates that in G. intraradices, expression of this MT gene is related to the levels of reactive oxygen species, as it was confirmed by the oxidative damage induced by paraquat in the extraradical mycelia, and suggests a role for GintMT1 in maintaining the fungal redox balance. Consistent with this protective function, MT synthesis is consistently induced by a variety of agents and conditions producing oxidative stress in different organisms (Bonneton et al. 1996; Murphy et al. 1999; Chen et al. 2004).

Because Cu is a redox-active metal that can be toxic through its participation in Fenton or Haber-Weiss reactions producing hydroxyl radicals (Halliwell and Gutteridge 1989), GintMT1 might be involved in maintaining the redox balance either by sequestering Cu and preventing deleterious Fenton reactions or by directly scavenging deleterious oxygen radicals. The finding that treatment of the mycelia with 5 mM Cu increased the level of lipid peroxidation 12 h after Cu addition whereas the induction of GintMT1 was not evidenced till 12 h later suggests a direct role for GintMT1 in scavenging reactive oxygen species under these experimental conditions.

Cd, a metal without redox properties per se, causes an increase in the levels of lipid peroxidation. Consistent with this observation, it has been reported in various organisms that Cd indirectly induces oxidative stress by depleting free-radical scavengers such as glutathione and protein-bound sulphydryl groups (Schutzendubel and Polle 2002). However, down-regulation of GintMT1 expression in both the Cd- and 50 μM Cu-treated mycelia suggests an alternative role for GintMT1, which does not necessarily involve HM sequestration or oxidative stress protection. Down-regulation of MT gene expression by various metals has been reported in different plant (Kawashima et al. 1991) and animal species (Costello et al. 2004) and has led to the hypothesis that MTs might be involved in the scavenging and transfer of HM to metalloproteins (Costello et al. 2004; Feng et al. 2005) or in stress signalling (Wong et al. 2004). Both of the above-described roles could be valid for GintMT1. In the case of acting as a metal scavenger, the inhibitions would be interpreted as the response to a situation in which the levels of cytosolic Cu rise and therefore, there is no need for a high affinity chelating system. Cd inhibition would be caused by its particular chemistry, which allows this element to mimic some bioelements such as Zn and Cu (Nath et al. 1984; Kaur et al. 2006). The other explanation suggests that GintMT1 inhibition would contribute to potentiate the generation of a free-radical-mediated signal, in a similar way than the rice MT OsMTb2 (Wong et al. 2004). Transient down-regulation OsMTb2, a reactive oxygen scavenger, during the oxidative burst phase during stress signalling is explained as a way to potentiate the accumulation of reactive oxygen species, which function as signals that trigger plant response to stress. However, with the available data, it is not possible to attribute a biological role to GintMT1 inhibition. Nevertheless and regardless of the explanation for the inhibition of GintMT1 transcription, it is clear that once a certain level of oxidative damage is reached, as evidenced by the level of membrane lipid peroxidation, GintMT1 transcription is enhanced.

In the plant pathogenic fungi Magnaportha grisea (Tucker et al. 2004) and Uromyces fabae (Jakupovic et al. 2006), MTs have been involved in the plant–fungus interaction. The MT-like protein NMT1 of M. grisea, which acts as a powerful antioxidant, is essential for appressorium-mediated penetration of intact leaf surfaces and plays a novel role in fungal cell-wall biochemistry that is required for fungal virulence (Tucker et al. 2004). Future studies on the regulation of GintMT1 along the life cycle of G. intraradices might shed some light on its role in the interaction of the AM fungus with its host plant.

In conclusion, all these data together suggest that GintMT1 might play a key role in the regulation of the redox status of the extraradical mycelia of G. intraradices through either its metal chelation activity or its thiol groups, which might contribute to the pool of cytosolic thiols that regulate fungal redox status.

Notes

Acknowledgements

This work was funded by the EU project Genomyca (QLK5-CT-2000-01319). Manuel González-Guerrero was supported by a FPU fellowship from the Spanish Ministry of Education, Culture and Sports. We are grateful to Dr. Maria Harrison for providing the GintMT1 partial clone and Dr. Simone Ottonello for the gift of the S. cerevisiae strain DTY113. We also want to thank Drs. José Miguel Barea and Alberto Bago for helpful discussions and Ascensión Valderas for excellent technical assistance.

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • M. González-Guerrero
    • 1
  • C. Cano
    • 1
  • C. Azcón-Aguilar
    • 1
  • N. Ferrol
    • 1
  1. 1.Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del ZaidínConsejo Superior de Investigaciones CientíficasGranadaSpain

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