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Genes related to heavy metal tolerance or homeostatsis (metallothionein, zinc transporter) in Glomus intraradices

Arbuscular mycorrhizal (AM) fungi are ubiquitous soil-borne microorganisms that establish a mutualistic symbiosis with most plant species. In AM symbiosis, the fungus supplies the plant with slow-diffusing nutrients, such as P, N, Zn and Cu, but they are also able to reduce metal translocation to the host plant when present at toxic levels in the soil. Consequently, AM fungi play an important role in protecting the host plant against heavy metal stress. Among the mechanisms of heavy metal tolerance, metallothioneins and metal transporters are of primary importance. Genes involved in metal homeostasis have been isolated from the AM fungus Glomus intraradices. Two cDNA fragments corresponding to a metallothionein (GintMT1) and Zn transporter (GintZnT1) were identified by exploring a G. intraradices EST library. The full-length sequences were obtained by RACE using gene-specific primers and cDNA from extraradical mycelium of the monoxenically grown fungus.

GintMT1 encodes a 71amino acid polypeptide with 13 Cys residues and a predicted molecular mass of 7.2kDa. It is related to gene members of the metallothionein family, showing 55% and 48 % similarity to the MT genes of the AM fungi G. rosea and G. margarita, respectively. Functional analysis of the gene through complementation of a yeast mutant defective in the metallothionein locus CUP1 showed in vivo protection of GintMT1 against Cd and Cu. Expression analysis of the gene through real time RT-PCR revealed down regulation of GintMT1 in the extraradical mycelium by a long-time exposure of G. intraradices to Zn, Cd or Cu. Sudden exposure of the extraradical mycelium to heavy metals or paraquat (an oxidative agent) showed up-regulation by high Cu concentrations and paraquat, but down-regulation by Zn and Cd. These data suggest the involvement of GintMT1 in the protection against oxidative stress, as well as in heavy metal homeostasis.

GintZnT1 encodes a 454 amino acid polypeptide with a predicted molecular weight of 54kDa. Based on its sequence and general structural features, GintZnT1 has been classified as a putative Zn transporter, included in the Cation Diffusion Facilitator (CDF) family of metal transporters. GintZnT1 shows the highest homology to the vacuolar transporters Zrc1 and Cot1 of Saccharomyces cerevisiae and Zhf of Schizosaccharomyces pombe. The predicted topology of the deduced amino acid sequence of GintZnT1 shows 6 transmembrane domains and two cytoplasmic motifs. All these regions are almost identical among GintZnT1 and the three previously characterized fungal CDF proteins. An effect of the GintZnT1 gene product on the cytoplasmic labile regulatory Zn pool of S. cerevisiae has been detected by using a Zn-regulated beta-galactosidase reporter gene, which indicates a role of GintZnT1 reducing cytoplasmic Zn availability. GintZnT1 expression in the extraradical mycelium of G. intraradices has been studied by using Real-Time RT-PCR. Up-regulation of GintZnT1 mRNA was detected upon short-time exposure of G. intraradices to Zn and when the mycelia were developed in 75 microM Zn supplemented plates. All these data suggest a role of GintZnT1 in Zn compartmentalization and in the protection of G. intraradices against Zn stress.

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Consejo Superior de Investigaciones Cientificas
Profesor Albareda 1
18008 Granada
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