Regulation of copper transporters in the arbuscular mycorrhizal symbiosisEffect on host plant copper homeostasis and development

Resumen

The transition metal copper (Cu) is a micronutrient acting as a redox active cofactor of key enzymes involved in a wide array of biochemical processes essential for life, such as respiration, superoxide scavenging, photosynthesis, ethylene perception and iron mobilization. However, when in excess, it becomes toxic due to its ability to displace other metal ions in structural or catalytic protein motifs and through the generation of hydroxyl radicals by Fenton-like reactions which can cause oxidative damage of DNA, lipids and proteins. Due to the dual nature of Cu, organisms have developed sophisticated homeostatic networks to tightly regulate Cu intracellular levels, in which membrane transporters mediating Cu uptake and efflux play a key role. Although Cu is usually present in trace amounts in soils, it can reach toxic levels in certain areas mainly due to anthropogenic activities, such as mining, soil overfertilization and the use of pesticides. Therefore, plants and in general all soil inhabitants have to deal with a wide range of environmental Cu concentrations, from scarcity to excess. One of the most prominent strategies that plant have evolved to overcome both situations, that is Cu scarcity and toxicity, is the establishment of mutualistic symbioses with arbuscular mycorrhizal (AM) fungi, soil-borne microorganisms belonging to the Glomeromycota. The fungal partner not only colonizes the root cortex but also maintains an extensive and highly branched network of hyphae that extends out the root beyond the depletion zone of nutrients, providing to the plant a new pathway, the mycorrhizal pathway, for the uptake and transport of low mobility nutrients, such as phosphorus, nitrogen, Zn and Cu. Besides improving plant mineral nutrition, AM fungi increase plant tolerance to biotic and abiotic stresses, such as salinity, drought and heavy metal contamination. The ability of arbuscular mycorrhizal fungi to benefit plant growth under Cu deficient and toxic conditions has been reported in many physiological studies. Different mechanisms have been proposed to explain the protective effect of the AM symbiosis in heavy metal stress. However, the mechanisms of Cu transport in the symbiosis are currently unknown. Within this PhD thesis, with the aim to get further insights into the mechanisms of Cu homeostasis in the AM symbiosis we have used a combination of in silico, physiological and molecular approaches, using the symbiosis established between the model AM fungus Rhizophagus irregularis, which is easily grown in monoxenic cultures and whose genome sequence is available, and different plant species. R. irregularis cultures were established in two experimental systems that allow to obtain exclusively fungal material for molecular analyses, in vitro in monoxenic cultures and an in vivo whole plant bidimensional experimental system. As a first step to further understand the mechanisms of Cu homeostasis in the fungal partner, a genome wide analyses was undertaken in order to establish a repertoire of candidate genes potentially involved in Cu transport. This in silico analysis allowed the identification in the genome of R. irregularis of seven open reading frames, which potentially encode Cu transporters, belonging to two multigene families. Three candidate genes belong to the CTR family of Cu transporters (RiCTR1-RiCTR3) and four to the P1B-ATPase family (RiCCC2.1-.3 and RiCRD). Comparison of these gene families with those of a set of reference fungi revealed an expansion of the R. irregularis CCC2 like-ATPases. Analyses of the published transcriptomic profiles of R. irregularis showed that RiCTR2 was highly expressed in mycorrhizal roots, suggesting that Cu is important for fungal colonization. These results are presented in Chapter I. To have some clues into the role of the R. irregularis CTRs in the symbiosis, we carried out the first functional characterization of the three putative sequences previously identified in its genome (RiCTR1, RiCTR2 and RiCTR3). These data are presented in Chapter II. We have shown that R. irregularis expresses two genes encoding Cu transporters of the CTR family, RiCTR1 and RiCTR2, and two alternative spliced variants of a third gene, RiCTR3. Functional analyses in yeast revealed that RiCTR1 encodes a plasma membrane Cu transporter and RiCTR2 a vacuolar Cu transporter. RiCTR1 was more highly expressed in the extraradical mycelia (ERM) and RiCTR2 in the intraradical mycelia (IRM). In the ERM, RiCTR1 expression was up-regulated by Cu deficiency and down-regulated by Cu toxicity. RiCTR2 expression increased only in the ERM grown under severe Cu-deficient conditions. These data suggest that RiCTR1 is involved in Cu uptake by the ERM and RiCTR2 in mobilization of vacuolar Cu stores. Cu deficiency decreased mycorrhizal colonization and arbuscule frequency, but increased RiCTR1 and RiCTR2 expression in the IRM, which suggests that the IRM has a high Cu demand. The third gene RiCTR3 produce two alternatively spliced products, RiCTR3A and RiCTR3B, highly expressed in the ERM. Up-regulation of RiCTR3A by Cu toxicity and the capability of its gene product to revert Cu sensitivity of the Δyap-1 yeast cells suggest that RiCTR3A might function as a Cu receptor necessary to activate downstream signal transduction pathways involved in Cu tolerance. Once that we showed that Cu uptake in R. irregularis ERM is mediated by the plasma membrane Cu transporter RiCTR1, whose gene expression increases under Cu deficiency but decreases under Cu toxicity, we tried to identify the transporter that could be involved in Cu transfer to the plant in the arbuscular interface. Export of metal ions takes place through PIB- type ATPases, also known as heavy metal ATPases (HMA), proteins that couple ATP hydrolysis to the transport of a heavy metal across different cellular membranes. The Rhizophagus irregularis genome has four candidate genes putatively encoding P1B-type ATPases. RiCCC2.1-3 are orthologs of the Saccharomyces cerevisaie CCC2 Cu-ATPase that transports Cu to Cu containing proteins in the trans-Golgi region and RiCRD1 is the ortholog of the P1B-ATPase CaCRD1 of the pathogenic yeast Candida albicans that exports Cu excess out of the cell providing Cu resistance. In Chapter lll, with the aim of determining whether RiCRD1 plays a role in Cu release to the apoplast of the symbiotic interface and/or in metal tolerance, the expression patterns of RiCRD1 were analyzed in the R. irregularis IRM and ERM developed in the presence of different Cu levels. Our results strongly suggest that RiCRD could be the transporter responsible for Cu release into the apoplast of the arbuscular interface, as our in situ hybridization experiments clearly revealed the presence of RiCRD transcripts in the arbuscules developed in the inner cortical cells of mycorrhizal tomato roots. On the other hand, RiCRD expression was up-regulated in the ERM by Cu and Cd toxicity. These results are consistent with a role for RiCRD in Cu and Cd detoxification by acting as a metal efflux pump, which agrees with its predicted localization at the plasma membrane. The higher induction of RiCRD expression in response to Cu toxicity than of other R. irregularis players of Cu tolerance, such as the metallothionein RiMT and the ABC transporter RiABC, suggests that RiCRD is the major determinant of Cu tolerance in R. irregularis. Here, we report for the first time a Cu efflux strategy to overcome metal excess in AM fungi. Overall, these data indicate that the Cu exporting ATPase RiCRD could have a dual role in Cu detoxification and symbiotic Cu nutrition, being, therefore, a key player of Cu homeostasis in R. irregularis. Finally, we have analyzed the effect of mycorrhizal inoculation on plant development and metal transport detoxifying mechanisms under Cu toxicity. In Chapter IV, we report how inoculation of a Cu sensitive cultivar of Zea mays with the AM fungus R. irregularis modifies the physiological plant response to Cu toxicity and HMA gene expression. Mycorrhizal plants presented a bigger biomass than non-mycorrhizal plants and less Cu concentration in their tissues. Interestingly, although some differences were found between the shoot nutrient content profiles of mycorrhizal and non-mycorrhizal plants grown under control conditions, these differences were higher in plants grown in soils supplemented with the highest Cu concentration. We found that the Zea mays genome harbors 12 genes putatively encoding HMAs, being ZmHMA3.3, ZmHMA5.1, ZmHMA5.2 y ZmHMA5.3 up-regulated under Cu toxicity. Interestingly, ZmHMA3.3 and ZmHMA5.3 expression was up-regulated only in roots and shoots of mycorrhizal plants. These data suggest that development of the symbiosis under Cu toxic conditions regulates plant Cu homeostasis, through the specific induction of certain proteins involved of Cu detoxification, potentiating in this way plant Cu tolerance. As the ZmHMA3.3 and ZmHMA5.3 gene products were predicted to be located at the tonoplast, up-regulation of these genes in roots and shoots of mycorrhizal plants suggests that, these transporters could play a dual role in shoot Cu protection of mycorrhizal plants by preventing root to shoot Cu translocation through its accumulation in the root vacuole and by vacuolar compartmentalization of the excess Cu that reaches the shoot. Overall, data presented in Chapter IV highlight the importance of the AM symbiosis in metal tolerance of their plant hosts. In conclusion, data presented in this thesis provide a great advance in the knowledge of Cu transport mechanisms in the AM symbiosis, especially in the identification and characterization of some Cu transporters of the AM fungus R. irregularis. However, further studies are necessary to completely understand how this complex network of protein transporters function in both the fungal and the plant partners.