- Top of page
- Materials and Methods
Heavy metal hyperaccumulation is an intriguing natural occurrence where plants can accumulate extraordinary quantities of metals into their shoots and leaves (Reeves & Brooks, 1983; Brooks, 1987), while storing lower concentrations in their roots, typically having root : shoot ratios < 1 (Baker, 1981; Macnair, 2003). Plants are generally considered to be hyperaccumulators at leaf concentrations of 1000 mg kg−1 for nickel (Ni), copper (Cu) and cobalt (Co), 10 000 mg kg−1 for Zn and Mn and 100 mg kg−1 for cadmium (Cd) (Baker et al., 1994; Brooks, 1998). There are approx. 400 species known to hyperaccumulate one metal or another, most of which are Ni hyperaccumulators, all of which occur on serpentine (Brooks, 1987). Serpentine floras and hyperaccumulators exist in several countries, such as New Caledonia, New Zealand, Albania, Greece, Italy and Cuba, and in California in the western USA (Brooks, 1987). A typical characteristic of serpentine soils is the abundance of potentially toxic heavy metals, particularly Ni and chromium. High magnesium (Mg) : calcium (Ca) ratios are also distinctive for these substrates and have been shown to be a factor in preadaptation to serpentine from granite (Taylor & Levy, 2002). Selection pressures associated with high amounts of Ni and other metals can result in the evolution of species and ecotypes that can safely accumulate and tolerate these metals (Kruckeberg, 1967).
Both Zn and Ni hyperaccumulation are more commonly studied than other metals, and some serpentine-adapted species are capable of hyperaccumulating both metals (Reeves & Brooks, 1983). Macnair (2003) suggests that metal hyperaccumulation could result from an enhanced efflux from the roots into the xylem. This suggestion corresponds to the results found by Lasat et al. (1998) who discovered that Zn had a five times greater concentration in the xylem of Thlaspi caerulescens compared with that of the nonaccumulating Thlaspi arvense. In comparison, Krämer et al. (1996) found a strong correlation between histidine and Ni accumulation where histidine concentrations were higher in the xylem of the Ni hyperaccumulator, Alyssum lesbiacum, than in the xylem of related nonaccumulators. Nickel and Zn are possibly transported by similar ligands and thus competition for binding sites is likely to be a factor in the relationship between uptake of Zn and Ni when levels of both are high in the soil (Assunção, 2003).
In addition to the interest in the physiological aspects of hyperaccumulation, such as metal uptake and transport (Krämer et al., 1996; Lasat et al., 1998; Persans & Salt, 2000; Assunção et al., 2001), there has been a considerable focus on the prospect of using hyperaccumulators for the phytoextraction of metals from contaminated land (Brooks, 1998; Salt et al., 1998; Escarréet al., 2000). In the exploration of phytoremediation and the potential of hyperaccumulators, it is important to know the extent of genetic variation within and between populations (Pollard et al., 2002).
The genetic variation between populations in their ability to hyperaccumulate Zn, Ni and Cd has been investigated in several studies using T. caerulescens populations from uncontaminated soils, as well as those from serpentine and calamine ones (Pollard & Baker, 1996; Escarréet al., 2000; Pollard et al., 2002; Assunção et al., 2003a). The results of these studies indicated a high level of variation for Zn, Ni and Cd accumulation between populations. A handful of studies have incorporated sib-analyses (Pollard & Baker, 1996; Meerts & van Isacker, 1997; Escarréet al., 2000; Macnair, 2002; Pollard et al., 2002) to study the quantitative genetics of the accumulation trait. All of these studies showed a degree of within-population genetic variation for Zn accumulation as well as variation for Ni (Pollard et al., 2002) and Cd (Escarréet al., 2000) accumulation within populations. Hitherto, this research strategy has been limited to two species, T. caerulescens and Arabidopsis halleri, both bodenvag species, Zn hyperaccumulators found growing on and off metalliferous soils. Thlaspi caerulescens, the focus of most of these studies, is considered to be a model hyperaccumulator (Assunção et al., 2003b). Many other Thlaspi species are also known to accumulate high levels of Ni and/or Zn (Reeves & Brooks, 1983; Reeves, 1992), yet these species have been far less studied despite the encouragement by Reeves & Brooks (1983).
Thlaspi caerulescens can hyperaccumulate Zn on several soil types, Ni on serpentine and Cd on Cd-rich soils, and there appears to be a diversity of transporter systems for different metals (Assunção, 2003; Assunção et al., 2003a). Assunção (2003) postulated that transporters involved with Ni hyperaccumulation are less metal-specific and are also responsible for low-affinity Zn and Cd accumulation in T. caerulescens populations from normal soils, serpentine (Ni-rich) and Cd-rich soils. She also suggested a high-affinity transporter system with high Zn preference in populations from different soil types: calamine (Zn/Pb-rich), nonmetalliferous (normal), Cd-rich, and Ni-rich (serpentine) (Assunção, 2003). Zinc hyperaccumulation has been considered a constitutive trait in T. caerulescens (Pollard et al., 2002; Assunção et al., 2003a). Thus, our goal was to determine whether two serpentine endemic species in Thlaspi, known to hyperaccumulate Ni in the field, have also evolved the Zn hyperaccumulation trait and to study the degree of genetic variation for Ni and Zn accumulation between and within populations.
We aimed to investigate differences between populations for Zn and Ni hyperaccumulation in Thlaspi pindicum, a Greek serpentine endemic (Tutin et al., 1993) and to examine genetic variation and heritability of hyperaccumulation in T. pindicum and Thlaspi alpinum var. sylvium, a species reported growing only on ultramafic substrates, located in Valle d’Aosta, Italy, and along the Swiss border (Reeves & Brooks, 1983; Verger, 1992). Meyer (1973) allocated both taxa to the Noccaea, the same section of the Thlaspi‘genus’ in which he located T. caerulescens. Thlaspi pindicum and T. alpinum var. sylvium are well-known Ni hyperaccumulators (c. 10 000 mg kg−1 Ni for both species), but Zn levels in herbarium specimens have been reported below 200 mg kg−1 for T. pindicum and ranging from 100 mg kg−1 to 3000 mg kg−1 for T. alpinum var. sylvium (Reeves & Brooks, 1983).
- Top of page
- Materials and Methods
The purpose of this study was to investigate, under standard conditions, the accumulation of Zn and Ni by two serpentine endemic taxa of Thlaspi, known from field data to accumulate Ni. We have shown that both taxa, T. pindicum and T. alpinum var. sylvium are capable of hyperaccumulating Zn under laboratory conditions although in the field they only accumulate Ni. There have been few other studies of this aspect of the hyperaccumulation phenomenon. Thlaspi oxyceras, a Turkish serpentine endemic and Ni hyperaccumulator, located in section Thlaspiceras (Meyer, 1973), has been shown to accumulate low levels of Zn – less than 1000 mg kg−1 from soils enriched with Zn and Ni (Peer et al., 2003). Species of Thlaspi in section Noccaea are more closely related to one another than they are to species in Thlaspiceras (Mummenhoff et al., 1997; Koch & Mummenhoff, 2001). Although T. oxyceras accumulates only low levels of Zn, other species adapted to serpentine in the Noccaea, such as Thlaspi ochroleucum, Thlaspi graecum, and Thlaspi epiretum have been found to accumulate higher levels of Zn (greater than 1000 mg kg−1, from serpentine) (Reeves & Brooks, 1983). By contrast, both the species studied here exhibited Zn and Ni accumulation patterns typical of the well-studied metal hyperaccumulator, T. caerulescens (Pollard & Baker, 1996; Meerts & van Isacker, 1997; Escarréet al., 2000; Pollard et al., 2002; Assunção, 2003). This species, which is found on both contaminated and uncontaminated soils, and on both calamine and serpentine soils, exhibits both Zn and Ni accumulation in all populations studied to date.
We found significant between-population variation for both Ni and Zn accumulation in T. pindicum. Populations from the Katara Pass, Metsovo Lake and the Peloponnese accumulated large quantities of Ni and Zn into their shoots, but these amounts were different among populations. The Katara Pass and Metsovo populations were superior to the Peloponnese population in that they accumulated the greatest amounts of Zn and Ni in their shoots, but only the Katara population was able to maintain root : shoot ratios below one in both Zn and Ni as well as the metals in combination. By contrast, we found no evidence of within-population genetic variation in two serpentine endemics, T. pindicum and T. alpinum var. sylvium. Ten maternal families of T. pindicum from the Katara Pass population as well as eight families in T. alpinum var. sylvium showed no significant variation for either Zn or Ni hyperaccumulation. Thus, no signs of genetic variation for metal hyperaccumulation and heritability for either metal could be detected. However, an interaction occurred between families and metal treatments for Ni accumulation for both species, indicating that there could be some genetic variation for the relative uptake of the two metals. There was also significant variation in the Zn root : shoot ratios, which could suggest that that there is variation in the mechanisms for metal translocation and transport. Thus, we have shown significant between-population variation in T. pindicum, but not significant within-population variation. This pattern is typical of a self-fertilizing species, but there has been no study of the breeding system of this species. Thlaspi pindicum is a close relative to T. caerulescens, which is primarily self-fertilizing, although the extent of this varies between populations (Dubois et al., 2003).
Our results can be compared with those of studies of the model hyperaccumulating species T. caerulescens, which is closely related to the serpentine endemics T. pindicum and T. alpinum var. sylvium (Meyer, 1973). Between-population variation in metal accumulation has often been found (Pollard & Baker, 1996; Bert et al., 2000; Escarréet al., 2000; Pollard et al., 2002; Assunção et al., 2003a; Roosens et al., 2003). Several genetic studies have also investigated within-population genetic variation for hyperaccumulation in this species (Pollard & Baker, 1996; Meerts & van Isacker, 1997; Escarréet al., 2000; Pollard et al., 2002). All of these studies have shown a small amount of genetic variation and some degree of heritability for Zn accumulation. Pollard et al. (2002) observed genetic variation among maternal families within three of five populations, including a serpentine population. Heritability estimates were determined to be above zero and some close to one, indicating that Zn and Ni accumulation varies in heritability in both metalliferous and nonmetalliferous populations.
In this study we also investigated the interaction between Zn and Ni accumulation when both metals are present in high concentrations. In all experiments, the accumulation of Ni was reduced when Zn was also present, while in two of the experiments the uptake of Zn was enhanced by the presence of Ni. Assunção (2003) also found that Zn was preferred over Ni in a serpentine population of T. caerulescens and she proposed that Ni hyperaccumulation is less metal-specific than Zn hyperaccumulation. Preference and competition between metals has previously been hypothesized for species growing in high concentrations of various metals (Boyd & Martens, 1998). In our study, competition is apparent between Ni and Zn for both species. The competition between the metals probably occurs in the roots since the Zn root : shoot ratios were not affected by Ni hyperaccumulation and the Ni root : shoot ratios were higher in the combined treatment.
If a serpentine endemic shows preferential accumulation of Zn rather than Ni, when it does not hyperaccumulate Zn in nature, then this might suggest that the processes leading to the evolution of Ni hyperaccumulation on serpentine are not solely or possibly even primarily for Ni accumulation. It is possible that Ni accumulation is the character under direct selection, but this has been achieved by acting on a pre-existing Zn accumulation system. Zinc accumulation would then be a residual effect of this character. Conversely, selection might be for an efficient metal uptake system capable of transporting and accumulating several different metals, as seen in T. caerulescens (Escarréet al., 2000; Lombi et al., 2000). Thlaspi caerulescens is known to hyperaccumulate Ni, Zn, Co, and Cd and can also accumulate lead (Pb) and manganese (Mn) to high levels (Baker et al., 1994). Nonetheless, because of the high Ni : Zn ratio in serpentine soil, such as in Table 1, an enhanced transport system with preference for Zn over Ni would perceivably result in higher Ni accumulation than Zn accumulation. This was evident with T. pindicum in nature (Table 2) but not T. alpinum var. sylvium, which accumulated more Zn in nature than Ni (Table 2), but the Ni concentration in the soil in the Valle d’Valtournenche was much less than that found in the Pindus Mountains (Table 1) where T. pindicum was collected.
The fact that these species appear to have the latent ability to hyperaccumulate Zn, though they do not in nature, would suggest that it would be difficult to postulate an adaptive hypothesis for this Zn accumulation phenotype. Adaptive hypotheses for hyperaccumulation have focused on herbivory deterrence, metal tolerance mechanisms, allelopathy and drought resistance (Boyd & Martens, 1992; Krämer et al., 1997; Pollard & Baker, 1997; Huitson & Macnair, 2003). Another hypothesis however, suggested by Boyd & Martens (1992), is that of inadvertent uptake, in which the phenotype is the byproduct of another process. Thus if some species of Thlaspi hyperaccumulate Zn inadvertently whilst foraging for another nutrient, then this would indicate that Zn hyperaccumulation has not evolved as a result of selection for that aspect of the phenomenon. If serpentine endemics preferentially hyperaccumulate Zn over Ni, then this might indicate that the translocation mechanism has a greater affinity for Zn than Ni. This would also suggest that Ni is transported via Zn transporters and thus Zn accumulation may have evolved first. Moreover, it appears that a high Zn affinity transporter system has evolved in species that grow only on Ni-rich substrates and may be present in most of the Zn and Ni accumulating species in Thlaspi. Further study is needed to focus on the inadvertent uptake hypothesis, which would shed light on why Zn hyperaccumulation occurs on nonmetalliferous soils as well as in serpentine endemics that rarely hyperaccumulate Zn in nature.