Plants have developed a variety of mechanisms to maintain the cellular concentration of essential metals within physiological limits. A complex homeostatic network controls the uptake, chelation and transport of metal ions to ensure healthy amounts of essential elements, and to avoid excess of non-essential ones (Williams and Salt, 2009). Metal transporters play a key role in metal uptake from the soil, metal homeostasis and metal transport across the cellular membranes. 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. Most members of the ZIP family fold into eight transmembrane domains with a variable metal-binding region, rich in histidine residues, between transmembrane domain III and IV (Guerinot, 2000). In Arabidopsis thaliana, 15 members of this family have been identified, and can be clustered in four main groups that share sequence similarity between 38 and 85%. To better understand the role of plant metal transporters, this project focuses on three ZIP proteins: AtZIP4, AtZIP6 and AtZIP9, for which limited information is available. AtZIP4 has been partially studied and the promoter analysis of this gene has been performed (Lin et al., 2016). Wintz et al. have shown that both ZIP4 and ZIP9, that share 77% similarity at the amino acid sequence, are up-regulated in roots and shoots of A. thaliana in Zn-deficiency conditions. On the contrary, AtZIP6, which doesn’t belong to any of the four groups mentioned above, does not show any modulation in expression at varying Zn or Fe concentrations. The promoter analysis for AtZIP6 and AtZIP9 has been performed on transgenic A. thaliana lines carrying the promoter sequence fused to the GUS reporter gene. The expression pattern of AtZIP6 and AtZIP9 showed different tissue localization. AtZIP9 expression is present in stomata in leaves, and localized in the root endodermis, cortex and epidermis, while AtZIP6 is expressed in the vascular tissues of both roots and shoots. Here, single AtZIP4, AtZIP6 and AtZIP9 knock-out mutant, zip4/zip9, zip4/zip6, zip6/zip9 double mutants and zip4/zip6/zip9 triple mutant obtained by crossing single homozygous mutant plants have been studied. The effect on growth of different Zn, Fe and Mn concentrations, and an ionomic analysis at different Zn concentrations, have been tested on these mutants looking for differences in the accumulation of multiple elements. To analyze the effect of the overexpression of ZIP4, ZIP6 and ZIP9, the coding sequences of the three genes have been cloned downstream of the strong promoter CaMV35S. The constructs were introduced into A. thaliana obtaining different over-expressing lines. Furthermore, the subcellular protein localization has been characterized in protoplasts transfected with genes encoding fusion proteins of ZIP4 and ZIP9 with GFP, and GFP observed at both the plasma membrane and tonoplast.
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