The genetic basis of tolerance and accumulation has been studied first through genetic approaches, analysing the segregating progenies of crosses between individuals with contrasting phenotypes. Interspecific crosses are sometimes required to study the hyperaccumulation phenomenon, which is found in different species of the same genus. One such example is the cross between A. halleri and A. lyrata (ssp. petraea) (L.) O'Kane & Al-Shehbaz, divergent for Zn and Cd hyperaccumulation. In the progeny, the traits of hyperaccumulation and tolerance segregate independently for both Zn (Macnair et al. 1999) and Cd (Bert et al. 2003).
Similar conclusions were reached by crossing T. caerulescens accessions differing for accumulation and tolerance to Zn (Assuncao et al. 2003) and Cd (Zha et al. 2004). In this specific case, non-tolerant plants accumulated more Zn than tolerant plants: this negative correlation can be explained by a pleiotropism of genetic determinants. In these experiments the conclusion is usually that tolerance and accumulation are regulated by one or few major genes, although they might interact in a pleiotropic fashion (Bert et al. 2003; Frerot et al. 2003). Segregating populations for Ni and Zn response demonstrated that for Ni, tolerance and accumulation are also independently inherited gene traits, whereas Ni and Zn accumulation are genetically correlated (Richau and Schat 2009). All authors have therefore concluded that metal hyperaccumulation cannot be considered simply a tolerance strategy. Similar approaches have been applied to search for co-segregation with specific genes. Hassinen et al. (2009) analysed segregation of allelic variants of genes for metallothioneins MT2a and MT3 in T. caerulescens. These alleles derived from the metallicolous parental accession had a higher level of expression, but this did not co-segregate with Zn accumulation in the progeny. Mutant analysis is a powerful instrument for genetics. The literature on metal tolerance and accumulation provides examples mainly for the model plant Arabidopsis thaliana (L.) Heynh. Mutants for metal tolerance can be selected by screening for increased or decreased survival at high metal concentrations. The selection of Cd sensitive mutants in A. thaliana have led to the discovery that one gene, phytochelatin synthase, was responsible for the synthesis of metal ligands (Ha et al. 1999). An A. thaliana T-DNA insertional mutant in the gene AtHMA4 shows a lower accumulation of Zn and Cd in leaves as compared to the wild-type plants; at the same time accumulation in roots is higher (Verret et al. 2004). In the same plants, mutants defective for HMA2 and HMA4 show increased sensitivity to Cd and a decrease in Cd shoot translocation; in the double mutant hma2, hma4 Cd translocation from roots to shoots was completely lost. Therefore, both HMA2 and HMA4 have a role in Cd translocation in A. thaliana (Wong and Cobbett 2009). Zinc-sensitive T-DNA insertional mutants of A. thaliana led to the identification of gene ZIF1, encoding for a tonoplast protein involved in vacuolar sequestration of Zn (Haydon and Cobbett 2007a). These mutants impaired in ZIF1 expression also show increased content of Zn in shoots, which is consistent with a reduced sequestration of Zn in the vacuoles. Caesium resistant mutants of A. thaliana have been isolated taking advantage of the collections of T-DNA insertional mutants (Marmiroli et al. 2009). Insertional mutants offer the possibility of cloning and identifying the mutated gene, but especially of identifying the putative functions involved in the phenotype. In this case, it was demonstrated with molecular and physical techniques that a single gene mutation impairs Cs uptake and translocation, as well as K and Ca homeostasis. Occurrence of mutants is often complicated by the redundancy of gene functions in plants. Guo et al. (2008b)
studied A. thaliana mutants defective in metallothionein genes MT1a and MT2b and found no decrease in metal tolerance. On the other hand, a triple mutant also defective for phytochelatin synthase is more sensitive to Cu and Cd as compared to the single mutants. Such experiments showed a role of metallothioneins in metal homeostasis to be possibly redundant with the role of phytochelatins.
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