Earlier studies on genes involved in plant metal tolerance and accumulation took advantage of the complementation of yeast mutants deficient in tolerance or in metal uptake. Zinc transporters (Grotz et al. 1998; Pence et al. 2000; Draeger et al. 2004), metallothioneins and metal-transporting P-type ATPase (Papoyan and Kochian 2004) were studied in this way. The availability of cloned genes allowed a series of interesting studies, as exemplified in the following sections.
Southern blot analysis with cloned genes can provide indications on the copy number. In A. halleri two genes are present in multiple loci as compared to other species. The metal transporter gene AhMTP1, homologue of A. thaliana ZAT, is triplicated in the A. halleri genome but is in single copy in the genomes of A. thaliana and A. lyrata (Draeger et al. 2004). The loci in A. halleri are not linked; they co-segregate with Zn tolerance and justify the high level of expression. Recent evidence showed that the A. halleri genome contains five copies of the MTP1 gene (Shahzad et al. 2010). The gene encoding for the P-type ATPase HMA4 is also triplicated in A. halleri with 99% sequence identity (Hanikenne et al. 2008) and quadruplicated in T. caerulescens (O Lochlainn et al. 2011). At variance with MTP1, the multiple copies of HMA4 are arrayed in tandem. In T. caerulescens, the gene IRT1 is duplicated: a full-length sequence and a truncated variant (Plaza et al. 2007) are expressed, respectively, in the Ganges ecotype (Cd accumulating) and in the Prayon ecotype (non-accumulating). The predicted structure of the gene products demonstrates that the shorter protein lacks a metal-binding site and five transmembrane helices, lacking the ability to transport Cd. The suppression of active Cd transport could be seen as an adaptive response to high metal concentrations.
Cloned gene sequences can be used to compare their expression patterns through northern blots, in species and individuals with contrasting phenotypes. Pence et al.
(2000) compared expression of the Zn transporter ZNT 1 in T. caerulescens and T. arvense, demonstrating that ZNT1 is highly expressed in roots and shoots in the hyperaccumulator, independently of the Zn supply. But the expression in the non-accumulator is up-regulated at low Zn concentrations. Metallothionein genes have been cloned and compared in ecotypes of T. caerulescens with different degree of Zn tolerance, showing higher expression of MT2 in shoots and roots of the Zn-tolerant accession (Hassinen et al. 2007). Tissue-specific expression can also affect the level of tolerance or accumulation. The ZNT1 gene of T. caerulescens has higher levels of expression in leaf cells, when compared to the A. thaliana homologue ZIP4, enhancing the difference in Zn accumulation in the aerial parts (Milner and Kochian 2008). Promoter efficiency can affect the level of tolerance and accumulation. This was demonstrated for HMA4 in A. halleri in transgenic plants: the promoter sequence of HMA4 from A. thaliana confers a lower level of transcription whereas the promoters from A. halleri are as effective as the cauliflower mosaic virus 35 S promoter (Hanikenne et al. 2008). Quite surprisingly, no attempt has been made to analyse the sequence of promoters in genes identified as relevant for metal tolerance and accumulation, dissecting their sequences and searching for consensus elements. Zientara et al. (2009) analysed the promoter of gene MRP3 from A. thaliana, describing its inducibility by Cd, Ni, As, Co, and Pb. However, this promoter does not come from a gene isolated from a tolerant or hyperaccumulator plant.
Sequences of cloned genes are available for bioinformatic analysis and alignment to construct phylogenetic trees, to search for variants in coding sequences, to search differences for those promoter sequences which may be related to differential gene expression. The alleles for ZNT1 cloned from the two accessions of T. caerulescens Prayon and La Calamine differ by 30 amino acids at the N-terminus and 5 amino acids inside the coding sequence (Assuncao et al. 2001). In a phylogenetic tree, ZIP transporters from angiosperms were compared, including ZNT and IRT genes (Plaza et al. 2007). Allelic variants among ecotypes of T. caerulescens are evidenced, pointing for a possible role in metal tolerance. To assess the importance of single amino acid residues, Song et al. (2004) compared the gene AtPcrs1, involved in tolerance to Cd, to other similar sequences present in A. thaliana and Oryza sativa L.. Modified gene sequences were checked for their capability in conferring Cd tolerance in mutant yeast. In this way, it was demonstrated that a complete Cys-rich region is important for the protein function in Cd tolerance. In T. caerulescens, the sequence of the MT3 gene for metallothionein shows differences in accessions with different tolerance and accumulation levels: the allele from a Cd tolerant accession has a modification in the Cys domain which can result in a larger cavity for chelation of metals (Hassinen et al. 2007). An interesting feature has been described for MTP1 in Thlaspi goesingense [now reclassified as Noccaea goesingensis (Halacsy) F.K. Mey.], a Ni hyperaccumulator:
two mRNAs have been found as a result of differential splicing of a genomic sequence (Persans et al. 2001). The two resulting proteins are supposed to have a role in sequestration of different metals inside the vacuoles. Alternative splicing has been described as a mechanism for regulating gene expression during heat shock (Sinibaldi and Mettler 1992), and it could be a mechanism for differential accumulation of metals. In the case of metallothionein genes cloned from different accessions of T. caerulescens, the alleles differ in the coding sequences, in the length of the 3' untranslated region and in the length of introns (Hassinen et al. 2009). In one cultivar of O. sativa identified as Cd accumulator, the phenotype has been attributed to a specific allele of gene HMA3, encoding for a vacuolar metal transporter (Miyadate et al. 2011). The allele carries a deletion in one exon due to variation in the number of tandem repeats, and the accumulator cultivar translocates higher amounts of Cd from roots to shoots as compared to other cultivars.
In silico analysis of coding sequences and of predicted amino acid sequences can provide indications about the putative functions and cellular localisation of the gene products. The coding sequence of the gene AhMTP1 in A. halleri contains a microsatellite region, a series of tandem repeats of a 6-bp sequence (CATGAT coding for HisGlu) whose length differs in the alleles from individual accessions (Draeger et al. 2004). The impact of this polymorphism on Zn tolerance has not yet been elucidated. The protein encoded by gene TcHMA4 has the presence of a C-terminal stretch rich in His and Cys residues which confers tolerance to Cd, presumably by providing binding sites for metal ions: tolerance is correlated to the length of this stretch (Papoyan and Kochian 2004). Concerning the subcellular localization of the proteins, the MTP1 gene in T. caerulescens is expressed in the tonoplast, whereas the homologue in T. goesingense, Ni accumulator, is expressed in the plasma membrane (Milner and Kochian 2008). However, a later paper also showed localisation of MTP1 to the tonoplast in T. goesingense (Gustin et al. 2009).
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