Functional characterisation of three zinc transporters in Thlaspi caerulescens

Heavy metal hyperaccumulation in plants is a poorly understood phenomenon. Transmembrane metal transporters are assumed to play a key role in this process. In the research described in this thesis, genes encoding Zn transporters of Thlaspi<span style='mso-bidi-font-weight:bold'>caerulescens</span><span style='mso-bidi-font-weight:bold;mso-bidi-font-style:italic'>, a heavy metal hyperaccumulator plant,</span>are studied and compared to their orthologues in Arabidopsis thaliana<span style='mso-bidi-font-style:italic'>, a non-hyperaccumulator plant</span>.<o:p></o:p></span>The Tc<span style='mso-bidi-font-style:italic'>ZNT1</span> and Tc<span style='mso-bidi-font-style:italic'>ZNT2</span> genes of T. caerulescens are members of the Zrt, Irt ( ZIP) -like gene family and show 89% and 87% similarity with the At<span style='mso-bidi-font-style:italic'>ZIP4</span> and At<span style='mso-bidi-font-style:italic'>IRT3</span> genes of A. thaliana<span style='mso-bidi-font-style: italic'>,</span>respectively. AtZIP4 and AtIRT3 genes are induced exclusively by Zn-deficiency in Arabidopsis. In limiting Zn conditions both Arabidopsis and Thlaspi show characteristic symptoms of Zn deficiency manifested both in vegetative and reproductive tissues. However, in T. caerulescens , Tc<span style='mso-bidi-font-style:italic'>ZNT1</span> and Tc<span style='mso-bidi-font-style:italic'>ZNT2</span> are constitutively and highly expressed in roots, irrespective of the Zn concentration in the medium. This observation suggested a role for<span class=GramE>these</span>genes in Zn uptake and possibly in explaining the Zn hyperaccumulation trait in Thlaspi . The proteins are localized in the plasma membrane, conferring zinc uptake into the cytoplasm. Tc<span style='mso-bidi-font-style:italic'>ZTP1,</span> another Zn transporter gene shows 85% sequence similarity in its coding region with the At<span style='mso-bidi-font-style: italic'>ZAT</span><span style='mso-bidi-font-style:italic'>/ MTP1 of</span>A. thaliana and is assumed to be localized in the vacuolar membrane. This gene belongs to the Cation Diffusion Facilitator (CDF) family.<o:p></o:p></span>The responses of the over-expression lines of TcZNT1,TcZNT2 and TcZTP1 in A. thaliana<span class=GramE>were</span>investigated with respect to deficiency and toxicity of Zn, Fe and Cd.</span><span lang=EN-GB style='font-family:"Times New Roman";mso-ansi-language:EN-GB'>Over-expression of Tc</span><span lang=EN-US style='font-family:"Times New Roman";mso-ansi-language: EN-US'>ZNT1</span><span lang=EN-GB style='font-family:"Times New Roman"; mso-ansi-language:EN-GB'>showed higher Zn accumulation in A. thaliana , whereas this effect was small and not significant for TcZNT2.TcZNT1 over-expression lines also showed increased Cd accumulation.</span><span lang=EN-US style='font-family:"Times New Roman";mso-ansi-language: EN-US'>In addition, the TcZNT1, TcZNT2 and TcZNT1/TcZNT2 silenced T. caerulescens showed</span><span lang=EN-GB style='font-family:"Times New Roman"; mso-ansi-language:EN-GB'>reduced Zn content in roots. These results, together with the plasma membrane localisation of TcZNT1 and TcZNT2 in cowpea protoplast, suggest a role in metal uptake of these two proteins in the plant. Over-expression of the TcZTP1 confers Zn and Cd tolerance and accumulation in A. thaliana. The regulation of expression of these genes was studied in comparison to the orthologous genes in A. thaliana by the study of the TcZNT1 and AtZIP4 promoter. The TcZNT1 promoter was isolated by PCR using forward primers designed on the A. thaliana gene upstream of AtZIP4 and a reverse primer on the T.<span style='mso-bidi-font-weight: bold'>caerulescens</span>TcZNT1 cDNA<span style='mso-bidi-font-weight:bold;mso-bidi-font-style:italic'>)</span>. The promoters of ZIP4 orthologues were isolated by a similar method from Arabidopsis halleri, Arabidopsis lyrata and Cochleria pyrenaica. The sequence analysis performed on the ZIP4 promoters shows the presence of two conserved palindromic sequences in all five species, suggesting that these are essential promoter elements for both the hyperaccumulators and non-accumulators. Using (deleted) promoter: GUS fusions transformed to A. thaliana , the cis elements of AtZIP4 were identified between</span><span lang=EN-US style='font-family:"Times New Roman"; mso-ansi-language:EN-US'>-263 bp and -115 bp from the start codon and</span><span lang=EN-GB style='font-family:"Times New Roman";mso-ansi-language:EN-GB'>for TcZNT1</span><span lang=EN-US style='font-family:"Times New Roman";mso-ansi-language:EN-US'>between -223 bp and -98</span><span lang=EN-GB style='font-family:"Times New Roman"; mso-ansi-language:EN-GB'>bp by 5' deletion study of the promoters. These regions also contain the two palindromic sequences in both promoters, indicating the importance of these conserved palindromes in the hyperaccumulators and non-accumulators. The AtZIP4pro-GUS transformed A. thaliana plants showed high expression in Zn-deficient roots, shoots, flowers and seeds. Similar expression pattern was observed in TcZNT1pro-GUS transformed A. thaliana plants. This suggests both the cis elements in the AtZIP4 and TcZNT1 promoters are conserved. To explain the high expression of TcZNT1 in T. caerulescens<span style='mso-bidi-font-style:italic'>,</span>the mode of cis/trans interaction and/or the expression profiles of the<span class=GramE>trans</span>genes in both species need to be studied. Most likely, the</span><span lang=EN-US style='font-family:"Times New Roman";mso-ansi-language:EN-US'>expression profile of the transcription factors and consequently many other genes functioning in metal uptake, transport and sequestration have been changed, in the hyperaccumulator plants as an adaptation strategy to survive in the highly contaminated soil. It is expected that the knowledge obtained can be used to design strategies to generate genetically engineered plants that can be used for phytoremediation and/or that have an improved nutritional quality