Functional analysis of AtZIP4, AtZIP6 and AtZIP9 metal transporters of Arabidopsis thaliana and Expression of Saccharomyces cerevisiae ZRC1 in different plant species.

Plants have developed a variety of adaptive strategies to take up sufficient quantities of essential macro- and micro-nutrients and avoid their excessive accumulation, which could be toxic. Metal transporters play an essential role in this homeostatic network by controlling the metal efflux across cellular membranes and compartments. The ZIP (ZRT IRT1-like Proteins: Zinc-regulated transporter Iron-regulated transporter 1-like protein) family of metal transporter is involved in this complex network. We have focused on three ZIP-family members: AtZIP4, AtZIP6 and AtZIP9, for which very little information is available. These genes have not been yet characterized although a possible role in micronutrients vascular system uploading and root uptake was hypothesized. ZIP6 is not part of the four main groups in the ZIP family and Milner et al., (2013) have highlighted its higher expression in roots than in shoots, whereas no expression modulation at varying Zn and Fe concentrations (Wintz et al., 2003). The transcript level of ZIP6 seems to be constitutively higher in the Zn hyperaccumulator Arabidopsis halleri ssp. halleri than in A. thaliana, independently of the Zn concentration (Becher et al., 2003). ZIP9 is clustered together with ZIP4, sharing c.a. 60% identity and 77% similarity at the amino acid sequence. Wintz et al. (2003) have shown that in A. thaliana both ZIP4 and ZIP9 are up-regulated in roots and shoots in Zn-deficient conditions. In A. halleri roots, ZIP9 is up-regulated upon Zn-deficiency and down-regulated in presence of high Zn concentrations. In A. thaliana shoots, ZIP9 level is detectable only upon excess Zn (Weber et al., 2004). The aim of this project is the functional analysis of AtZIP6 and AtZIP9, to understand their expression pattern and their subcellular localization. AtZIP6 and AtZIP9 knock-out mutant lines were investigated and will be used for testing metal tolerance and accumulation in order to identify which metal(s) are transported by these proteins. The high similarity between ZIP9 and ZIP4 prompted us to add AtZIP4 mutant our analysis to avoid a possible functional complementation. Furthermore, over-expressing A. thaliana lines have also been obtained fusing the 35S constitutive promoter to the AtZIP6 and AtZIP9 coding sequence in order to perform a phenotypic characterization and a metal accumulation analysis in comparison to single zip4, zip6, zip9, double zip6/zip9, zip4/zip9, zip4/zip6 and a triple zip4/zip6/zip9 knock-out mutants and wild-type lines Another part of the project concerns the effect of heterologous yeast genes in A. thaliana, tobacco and poplar plants, analyzing their ability in heavy metal accumulation. ZRC1 gene was selected since it induces Zn/Cd resistance in Saccharomyces cerevisiae. ScZRC1 is a vacuolar transporter that mediates the detoxification of Zn excess storing it into the vacuole (MacDiarmid et al., 2003). ScZRC1 belongs to the CDF transporter family and might have also a putative role in Cd detoxification.