To better dissect some of the genetic features of the hyperaccumulator phenotype, comparative genetic and molecular analyses between hyperaccumulator and non-hyperaccumulator phenotypes are of primary interest (Fig. 3). It is essential that the genetic and molecular comparison involves species with identical or similar genetic background. In some cases, the analysis of the interspecies variability has allowed the detection of features related with hyperaccumulation. It was from the interspecific cross between the hyperaccumulator A. halleri and the non-hyperaccumulator Arabidopsis lyrata that some interesting QTLs for hyperaccumulation were detected (Bert et al. 2002, 2003). Transcriptomic studies were also performed at interspecies level with microarray and real-time PCR technologies comparing A. thaliana and phylogenetically related hyperaccumulator species such as A. halleri or T. caerulescens, leading to the identification of genes specifically modulated in hyperaccumulators (Weber et al. 2004; Becher et al. 2004; Rigola et al. 2006; van de Mortel et al. 2006, 2008). But the potential of these methodologies alone is not sufficient because of the different level of genomic information between A. thaliana and the hyperaccumulator species. Genomic sequences of hyperaccumulators are very limited and this impedes the identification of unique gene functions.
At the proteomic level, the comparison of two different species can also be difficult. Attempts were performed with the proteome of the Ni hyperaccumulator A. lesbiacum with that of a related non-accumulator species, Alyssum montanum, in the absence of Ni. Unfortunately, the 2D-PAGE protein patterns of the two species were not similar enough to allow a quantitative comparative analysis (Ingle et al. 2005). Therefore, comparative proteome analysis seems more suitable for
Fig. 3 Genomic and proteomic strategies proposed for molecular analyses in hyperaccumulators. Comparative proteomic approaches can be coupled with DNA and transcriptomic analyses to study the genotypic and phenotypic variation within and between species, thus providing useful DNA and protein markers for the elucidation of hyperaccumulator phenotypes
Fig. 3 Genomic and proteomic strategies proposed for molecular analyses in hyperaccumulators. Comparative proteomic approaches can be coupled with DNA and transcriptomic analyses to study the genotypic and phenotypic variation within and between species, thus providing useful DNA and protein markers for the elucidation of hyperaccumulator phenotypes individuals within the same species adapted to grow in different environments, where it is possible, such as in the case of T. caerulescens where genetic variation within the species has been well documented (Assuncao et al. 2003a, b; Yang et al. 2005; Richau and Schat 2009) (Fig. 3). Inter-population comparisons at transcriptomic and proteomic levels can be carried on avoiding the drawback due to inter-species variation, and could help in the identification of possible gene/ protein biomarkers for hyperaccumulation of a specific metal. In the work of Toumainen and collaborators (2006), comparative proteomic analyses were performed on three T. caerulescens populations LC, LE and MP considering not only different metal stress conditions but also plant accessions differing in tolerance and hyperaccumulation for Zn and Cd. Many differences were also seen among the Thlaspi accessions in absence of metal treatments. Proteomic variations between populations of the same hyperaccumulator species T. caerulescens were found in another work by comparing the metallicolous MP population and the non-metallicolous RpR population (Visioli et al. 2010b). The inter-population variation observed in both cases was not unexpected, because the different Thlaspi accessions grow naturally in different environments, and have other phenotypic differences. Selection by environmental conditions can lead to local adaptation and to differentiation of sub-populations in quite a short time, which can eventually also result in changes in protein patterns (David et al. 1997). In addition, in the specific metalliferous environment, the plant proteome is not only affected by metal concentration but by multiple environmental factors like: light, heat and water which all interfere with the metal stress response, the metal accumulation and the metal tolerance. The thesis in this case is that for any primary stress there are also both a secondary and a tertiary stress, and all contribute to establish the global phenotype (Levitt 1980).
Until now, most of the proteomic work performed refers to laboratory conditions in which the metal is the only stressor. Differences were found in the accumulation capacity when comparing the same plant accession grow in laboratory conditions and in natural soil. T. caerulescens LC which is a Cd hyperaccumulator in its soil of origin, in laboratory conditions shows a great Cd tolerance but a lower hyperaccu-mulation capacity than in the soil of origin (Tuomainen et al. 2006). Studying the relationships between plant and environment can help in elucidating important protein functions putative for the hyperaccumulator phenotype. Comparing pheno-typic variation within the Ni hyperaccumulator T. caerulescens MP population grown in its peculiar environment, it was found that the hyperaccumulation capacity is affected not only by soil composition but also by different micro-environmental conditions. These different accumulation capacities within population is reflected in abundance of specific sets of proteins related to metals, as metal transporters, and proteins related to defence against biotic and abiotic stresses and proteins of general metabolism (Visioli et al. unpublished data).
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