The root system represents the gate a metal had to cross to enter into the plant. Enhanced metal uptake combined to a reduced sequestration in the root vacuoles and the high translocation diversify hyperaccumulators and non-hyperaccumulator species (Verbruggen et al. 2009; Maestri et al. 2010). In roots, many Zn-transporters including ZNT1 are Zn-regulated in non-hyperaccumulators, e.g. they are only detectable under Zn deficiency, but in hyperaccumulators, they are constitutively expressed, independently of Zn supply (Pence et al. 2000; Assuncao et al. 2001). The iron transporter IRT1 has shown to also be correlated with the transport of Cd and Zn in the hyperaccumulator T. caerulescens (Lombi et al. 2002). Proteomic studies on root systems have been performed in the Ni hyperaccumulator Alyssum lesbiacum (Ingle et al. 2005) and in the Cd/Zn hyperaccumulator T. caerulescens (Tuomainen et al. 2006, 2010) (Table 1). In the former work, proteins that increase in abundance after a short, high Ni treatment (0.3 mM) were involved in sulfur metabolism, protection against reactive oxygen species (ROS) and heat-shock (HS) response. Few modifications were observed in the root system after a prolonged treatment with a 0.03-mM Ni concentration which is considered optimum for growth of this species but enough to trigger hyperaccumulation. Under this latter Ni treatment, constitutively expressed genes and proteins in high Ni-tolerant species may allow for effective chelation and sequestration of Ni without the need of further protein synthesis. In the work of Tuomainen and collaborators (2006) the roots proteome of three different T. caerulescens accessions [Zn/Cd tolerant La Calamine (LC), and Zn/Cd hyperaccumulator but less tolerant Lenningen (LE), and Monte Prinzera (MP)] was compared after 3-week treatments at various Zn and Cd concentrations. Different classes of proteins whose abundance changes between metal exposure but also between accessions were identified. Proteins related to removal of ROS were more abundant in the more hyperaccumulator but less tolerant accessions MP and LE while showing a lower level of abundance in the less hyperaccumulator but more tolerant LC accession. The enzyme superoxide dismutase (SOD), which is a Zn-requiring enzyme, decreases in Zn deprivation conditions in the LC and MP. A decrease in ascorbate peroxidase (APX) in root at high metal concentrations was also observed in the same accessions. The authors postulate that these modifications could be due to an increase in ROS leading to increased lignifications and in different metal binding and uptake capacity.
Root cell walls can also be influenced by metal stress. Synthesis of a protein related to cell wall structure, a putative glycosyl hydrolase family of 18 proteins, showed a modulation according to accession and treatment. This protein which participates in the re-assembling of cell wall and particularly in cell expansion was less abundant in the roots of MP accession which effectively accumulate Cd and Ni in the roots rather than in the LC accession. The root cytosolic glutamine synthe-tase, which is specifically expressed in root pericycle and is involved in ammonium assimilation, is known to be inhibited by Cd, causing ammonium accumulation and toxicity. The presence and abundance of this protein was correlated with Cd tolerance rather than to Cd accumulating capacity in the three accessions. Tuomainen and collaborators (2010), using a proteomic approach on a segregating population of the cross between T. caerulescens LC and LE accessions demonstrated that proteins whose abundance co-segregates with Zn accumulation in the F3 progeny are, for the greatest majority root proteins. Therefore, differences in Zn accumulation between the two accessions were mainly determined by the root proteome. In A. halleri, the transcript of HMA4 gene, one of the genetic determinants of the hyperaccumulator phenotype, is also preferentially expressed in the roots (Hanikenne et al. 2008). Furthermore in the hyperaccumulator T. caerulescens the concentration of histidine, which contributes to metal (Zn and Ni) loading, is enhanced in roots but not in shoots as compared with the non-hyperaccumulator Thlaspi arvense (Richau et al. 2009). Protein,s, transcript,s and metabolite,s concentrations were all more abundant in roots of hyperaccumulators suggesting a specific function according to the literature data. Proteome research regarding root tissues has failed to identify root transporters differentially abundant between treatments or between hyperaccumulator and non-hyperaccumulator plants. These data are in accordance with transcriptomics analyses which showed a constitutive expression of metal transporter genes in hyperaccumulators (Assuncao et al. 2001; Weber et al. 2004; Hammond et al. 2006). On the other hand it cannot be excluded that a particular transporter protein cannot be identified after the two-dimensional separation phase, due to some technical limitation of the proteomic approach (Rose et al. 2004).
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