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Nickel (Ni) hyperaccumulator plants represent over three-quarters of known metal hyperaccumulators, with a total number of 318 taxa reported to date (Baker et al., 2000; Reeves & Baker, 2000). Recently, Berkheya coddii has attracted attention because of its high Ni concentration and rapid biomass production. Robinson et al. (1997) reported an annual biomass production of 22 t ha−1 and up to 1% (w : w) Ni in the dry above-ground biomass. Berkheya coddii is an asteraceous summer-green perennial plant that is found on ultramafic (serpentine) soils in southern Africa (Morrey et al., 1992).
The phenomenon of Ni hyperaccumulation by plants is not well understood. Krämer et al. (1997) reported that at low substrate Ni concentrations the rates of Ni uptake and root-to-shoot translocation in both the hyperaccumulator Thlaspi goesingense and the nonNi-hyperaccumulator Thlaspi arvense were similar. It was suggested that Ni tolerance alone could explain Ni hyperaccumulation when plants were grown in presence of large concentrations of available Ni. More is known in terms of Ni tolerance in hyperaccumulator plants where two mechanisms, complexation and compartmentalization, appear to be responsible for Ni detoxification. The complexation of Ni by histidine and organic acids such as citrate, malate and malonate has been reported in a number of hyperaccumulator plants (Lee et al., 1977, 1978; Krämer et al., 1996; Brooks et al., 1998). Similar to what has been observed in cadmium (Cd) and zinc (Zn) hyperaccumulation, Ni has been reported to accumulate in the epidermal cells in Senecio coronatus and several species of Alyssum (Mesjasz-Przybylowicz et al., 1994; Psaras et al., 2000). In particular, Küpper et al. (2001) showed that Ni was preferentially compartmentalized in the vacuoles of epidermis cells in T. goesingense, Alyssum bertolonii and Alyssum lesbiacum. Using cell fractionation, Krämer et al. (2000) also showed that intracellular Ni is predominantly localized in the vacuoles of T. goesingense. This pattern of cellular distribution is consistent with the finding by Persans et al. (2001) who reported a high-level expression of a vacuolar metal ion transporter protein (TgMTP1) in T. goesingense. This transporter may account for the enhanced accumulation of Ni in leaf vacuoles of T. goesingense. Most of the information regarding the mechanism of Ni hyperaccumulation has been obtained in two genera of the Brassicaceae: Thlaspi and Alyssum. Very little is known about Ni hyperaccumulation in plants belonging to other families. Therefore, it is not known whether the same mechanisms are shared by all Ni hyperaccumulators.
In this paper, we investigate some aspects of Ni hyperaccumulation in B. coddii because of its high biomass, high Ni accumulation and its consequent potential to be used for the phytoextraction (either phytoremediation or phytomining) of Ni. Compartmentalization of metals seems to be a key mechanism of tolerance in hyperaccumulator plants (for a recent review see McGrath et al., 2002). Therefore, the aim of this study was to determine the distribution of Ni and other elements, at whole plant and cellular scale, in B. coddii.
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- Materials and Methods
Berkheya coddii showed all the characteristics typical of hyperaccumulator plants: it did not display phytotoxicity symptoms when exposed to elevated Ni concentrations (up to 500 µm); Ni concentrations in the plants were well above the 1000 µg g−1 reported as the lower limit for Ni-hyperaccumulation (Brooks et al., 1977); the concentration of Ni in the shoots was higher than in the roots. The results obtained confirm the elevated tolerance of this plant to large external and internal Ni concentrations and its efficient transport of Ni from roots to shoot.
Most Ni accumulated by B. coddii (71%) was found in shoots (Fig. 1). Küpper et al. (2000) and Zhao et al. (2000) suggested that the apparent metal accumulation in roots of hydroponically grown plants may be partly due to metal precipitation in the apoplast. In our experiments, EDXA analyses of B. coddii roots from the 500 µm Ni treatment showed high concentrations of Ni and P on the surface of the rhizodermis (data not shown), consistent with the suggestion that Ni is present as a phosphate precipitate. Hyperaccumulator plants usually store more metal in the shoots than in the roots. For example, 70–80% of the total Zn hyperaccumulated by Arabidopsis halleri and Thlaspi caerulescens was reported to be stored in the shoot (Shen et al., 1997; Perronnet et al., 2000; Zhao et al., 2000).
Within the shoot, leaves appeared to be the primary sink for Ni in B. coddii (Fig. 1). Another characteristic that seems to be common to metal hyperaccumulator plants is the fact that metals are largely stored in water-soluble forms. Zhao et al. (2000) reported that 60–65% of the shoot Zn in A. halleri was water-soluble. In this study, we found that most Ni (65%) in the leaves was extractable with water at pH 6.
At the shoot level, the concentration of Ni in the leaves decreased from older to younger leaves whereas Ni concentration in the corresponding parts of the stem followed an opposite trend, with the larger concentration of Ni found near the apex (Fig. 2). The distribution pattern of nickel in B. coddii indicates that transpiration has a large influence on the long-distance transport of within plants.
The concentrations of K, Fe and Cu in the shoot of B. coddii were significantly reduced by increasing Ni concentrations in the hydroponic solution (Table 1). In the roots, however, there was no significant difference in the concentration of these elements between the treatments. This may indicate that the uptake of K, Fe and Cu was not affected, but their root-to-shoot translocation was reduced when Ni concentration in the nutrient solution increased. However, increasing Ni in the nutrient solution decreased Ca concentrations in all parts of the plants. This may indicate a competition between Ca and Ni for plant uptake. Zhao et al. (2000) reported that the concentrations of K, Ca, P, Fe, Mn, Cu and Ni decreased in the shoot of A. halleri when the plants where exposed to increasing concentration of Zn. Similarly, Wenzel & Jockwer (1999) reported that K translocation from roots to shoots was depressed in Ni hyperaccumulators when grown in high-Ni substrates.
Accumulation of metals in an intracellular compartment, most likely the vacuole, of epidermis cells seems to be a common feature in hyperaccumulator plants (for a review see McGrath et al., 2002). Preferential accumulation of Ni in the intracellular compartment of epidermis cells of A. bertolonii, A. lesbiacum and T. goesingense has been reported by Küpper et al. (2001). However, most of the hyperaccumulators in which metal compartmentation has been investigated are brassicaceous. In the asteraceous B. coddii, we found significantly higher Ni concentrations in the apoplast of the upper epidermis cells, especially within the cuticle (Figs 3 and 4). This pattern of cellular distribution of Ni resembles that of Al in mature leaves of tea (Camelia sinensis; Matsumoto et al., 1976; Memon et al., 1981) where Al was found to be localized in cell walls and cuticle. In the case of Ni, however, a lot of metal was found inside the cells, whereas Al was exclusively outside the cells. Mesjasz-Przybylowicz et al. (2001) studied the distribution of Ni in young apical leaves of B. coddii using a nuclear microprobe and freeze-dried samples. They found that the highest Ni enrichment occurred in leaf margins, mesophyll and midrib epidermis. However, these results are difficult to compare with our findings because we used frozen hydrated samples and mature leaves. In dried samples, cellular compartmentation of elements may have been affected because metals may be redistributed in the fixation and drying processes (van Steveninck & van Steveninck, 1991).
The Ni distribution in B. coddii differs significantly from what has previously been reported for other Ni hyperaccumulator species, where this metal appeared to be compartmented mainly in vacuoles. Vacuolar sequestration of Ni and other heavy metals has been proposed as a key tolerance mechanism in hyperaccumulator plants (Krämer et al., 2000; Küpper et al., 2001; Persans et al., 2001). In the case of B. coddii, compartmentalization of Ni in the upper cuticle may help protect the cytoplasm from toxic concentrations of Ni. The disproportionately higher cuticle Ni concentrations in the 500 µm Ni treatment compared with the 100 µm Ni treatment may indicate that nickel is translocated to the cuticle when a threshold concentration in the living tissues is exceeded. However, the biological importance of cuticle-bound Ni is unclear because the total mass of Ni that is stored in the cuticle is likely to be small compared with the Ni contained in the leaf. This is due to the small total biomass of the cuticle.
Most other hyperaccumulator plants store heavy metals in the vacuoles of epidermal cells. High metal concentrations in the epidermis may indicate a predator defence role for the metal, as these are the first tissues encountered by grazing animals. However, the high Ni concentrations in the upper, but not the lower cuticle are not consistent with a predator defence role for Ni because many insect herbivores feed on the underside of leaves. Another possible role for nickel in the upper cuticle may be as a defence against ultraviolet radiation and protection of the underlying chlorophyll from solarization. This could be tested by growing plants under elevated levels of ultraviolet radiation in the presence and absence of Ni.