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- Materials and Methods
To survive, plants must supply thousands of transition metal-requiring apo-proteins in different locations with adequate amounts of the respective required transition metals and store excesses or nonessential transition metals in innocuous forms. Because of the high similarity between the ligand-binding preferences of divalent cations of different transition metal elements, this is a demanding task in biology, the operation of which is still poorly understood. The maintenance of metal homeostasis is particularly challenging for organisms in metal-rich environments, where plants are likely to experience episodes of exposure to large excesses of specific metal ions in the soil solution (Krämer et al., 2007). The aim of this study was to investigate the interaction of an excess of nickel (Ni) with the homeostasis of copper and iron in plants. This work was conducted using the nickel hyperaccumulator plant Alyssum inflatum originating from Ni-rich serpentine soils of western Iran (Ghaderian et al., 2007).
Metal hyperaccumulation is a characteristic of a small proportion of metallophyte and pseudometallophyte taxa, which can accumulate metals in their aboveground tissues in concentrations that are highly toxic for other plants (Baker et al., 2000). Nickel hyperaccumulator species grown in their natural habitats contain more than 1000 µg Ni g−1 shoot dry biomass and up to 47 500 µg Ni g−1 shoot dry biomass (Kelly et al., 1975; Baker & Brooks, 1989). In general, Ni hyperaccumulators are mostly found on serpentine soils which are naturally rich in the transition metals Ni, iron (Fe), cobalt (Co) and chromium (Cr) (Baker & Brooks, 1989). Identifying and developing plants suited for environmental technologies such as phytoremediation and phytomining requires a better understanding of metal homeostasis especially in metal hyperaccumulator plants, which are candidate species for use in these technologies or may serve as models for their development. In addition, the identification and physiological characterization of novel metallophyte species are crucial to exploring and preserving their biodiversity (Whiting et al., 2004).
At the physiological and molecular levels, the mechanisms that govern Ni hyperaccumulation and associated hypertolerance are not well understood. One of the characteristics of Ni hyperaccumulator plants in the genera Alyssum and Noccaea is their high chelator content, in particular free histidine, which has an important role in the high root-to-shoot Ni flux associated with Ni hyperaccumulation, as well as in Ni hypertolerance (Krämer et al., 1996; Kerkeb & Krämer, 2003; Wycisk et al., 2004; Ingle et al., 2005a; Richau et al., 2009). The high levels of histidine in Alyssum hyperaccumulators are a consequence of constitutively high expression of genes encoding rate-limiting enzymatic steps in the biosynthesis of histidine (Ingle et al., 2005a). In addition, increased glutathione biosynthesis has been observed in Thlaspi Ni hyperaccumulator species (Freeman et al., 2004), which appears to be a consequence of enhanced salicylic acid signals (Freeman et al., 2005). In the shoots of Alyssum Ni hyperaccumulators, the highest Ni concentrations have been observed in trichomes and epidermal cells (Krämer et al., 1997a; Broadhurst et al., 2004), and, quantitatively, leaf vacuoles have a prominent role in Ni storage (Ingle et al., 2008). In order to develop additional hypotheses about the physiological and molecular mechanisms of Ni hypertolerance it is necessary to better understand how Ni toxicity arises.
As an essential element, Ni is required in plants in small amounts (Brown et al., 1987; Dalton et al., 1988; Marschner, 1995), whereas much larger amounts are required of Fe, Zn and copper (Cu). One of the well-known symptoms of heavy metal toxicity in plants is oxidative stress (Dietz et al., 1999; Clemens et al., 2002; Clemens, 2006; Krämer & Clemens, 2006; Seregin & Kozhevnikova, 2006). Exposure to high concentrations of Ni was reported to cause an increase in the accumulation of proteins in sulphur metabolism and protection against reactive oxygen species (ROS) and heat-shock responses (Ingle et al., 2005b). Nickel toxicity symptoms described in plants include interveinal chlorosis, inhibition of root growth and induction of potassium (K) leakage (Woolhouse, 1983; Gabbrielli et al., 1989).
In addition, reports of the amelioration of Ni toxicity symptoms by calcium (Ca) or Fe fertilization suggest that the toxicity of Ni might result from its interference with Ca or Fe nutrition (Woolhouse, 1983; Gabbrielli & Pandolfini, 1984). Generally, the Irving–Williams series (Zn2+ < Cu+ > Cu2+ > Ni2+ > Co2+ > Fe2+ > Mn2+ > Mg2+ > Ca2+) predicts that in a given metal–ligand complex, a divalent metal cation can substitute all metal cations positioned downwards (da Silva & Williams, 1991). Accordingly, excess Ni can be predicted to interfere with the homeostasis of several other metals, most prominently that of Fe, which is required in the greatest amounts of all transition metals because of its critical roles in abundant proteins of photosynthesis and respiration, as well as in antioxidant activities and in numerous metabolic pathways (Marschner, 1995). Furthermore, excess Cu is predicted to interfere with Ni homeostasis.
The results presented here suggest that growth in high, subtoxic Ni concentrations specifically enhances Cu accumulation and Cu sensitivity of A. inflatum. Nickel toxicity in A. inflatum is at least partly a consequence of a deterioration of Fe-dependent protein functions in the shoots resulting from Ni-mediated disruption of Fe partitioning from roots into shoots.