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- Materials and Methods
Hyperaccumulation is a fascinating phenomenon in which a very small class of species accumulate exceptionally high concentrations of metals in their aerial parts (Brooks, 1998). With only very few exceptions, hyperaccumulation appears to be a species level trait, with all populations and individuals of a species exhibiting the character. There is substantial interest in the phenomenon because of the potential to use hyperaccumulating plants to phytoremediate land (Salt et al., 1998), and some progress is being made in understanding the mechanisms and physiology of the character (Persans & Salt, 2000; Assunção et al., 2001; Clemens, 2001).
In the field, individual plants of a hyperaccumulating species exhibit a very wide variation in phenotype, even within a single population (Bert et al., 2002). For instance, in the zinc hyperaccumulator Arabidopsis halleri, plants sampled from contaminated sites within the Harz mountains, Germany, had concentrations of leaf zinc of 0.6–5% zinc on a d. wt basis (unpublished data). It is important to know what factors lead to this variation. Intuitively, two of the most important determinants could be bioavailable soil zinc concentration and individual genotype. It is expected that soil zinc levels should be related to the plant metal concentration, although the relationship may not be linear, and it is possible that plant metal concentration could be relatively insensitive to soil metal concentration over a wide range of soil metal levels if the curve relating plant metal concentration to soil concentration saturates at quite a low external zinc concentration (Baker, 1981). Clearly genetic variation in accumulating ability could lead to variation in plant metal concentration.
There have been only a few studies of genetic variation in this character, and the majority of work has been conducted on the zinc/nickel hyperaccumulator Thlaspi caerulescens. A number of studies have found that different populations of this species exhibit variation in tolerance and accumulation of zinc, and also in the ability to accumulate zinc in above ground tissues (Ingrouille & Smirnoff, 1986; Pollard & Baker, 1996; Meerts & Van Isacker, 1997; Escarréet al., 2000). Pollard & Baker (1996) studied two populations of T. caerulescens from Britain. They found that there was significant interpopulation variation in zinc accumulation, and one of the populations showed significant genetic variance for this character. Meerts & van Isacker (1997) compared metallicolous and nonmetallicolous populations of T. caerulescens. They found that metallicolous populations were more tolerant of zinc, and accumulated less zinc in aerial parts. They also found substantial variation between families within populations, suggesting heritable variation for this character. Further studies on this species have found that different accessions differ in their relative accumulation of different metals (Lombi et al., 2001; Assunção et al., 2001).
Less work has been conducted on A. halleri.Bert et al. (2000) studied zinc accumulation in two populations of A. halleri. They found that the population from the uncontaminated site accumulated more zinc than the one from a contaminated site. Bert et al. (2002) studied the zinc concentration of field collected plants from a number of contaminated and uncontaminated sites. They found wide variation in zinc concentration, which was not easily reconciled with the contamination status of the sites. They did not, however, test the ability of the plants to accumulate metal, which can only be done under standard conditions. In this paper, I investigate the accumulation of zinc by a number of populations of A. halleri from both contaminated and uncontaminated environments, and test the hypothesis that accumulation is related to soil contamination. In addition, I test whether there is genetic variation within populations for zinc accumulation.
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I have shown that there is considerable variation both between and within populations in the ability to accumulate zinc under standard laboratory conditions. In experiment 1, plants accumulated zinc steadily for about 7 wk until they had reached a value typical of field collected plants. The rate of accumulation, and final concentration accumulated, was not particularly affected by the external concentration. The plants showed extensive variation in final zinc concentration. In experiment 2, a range of populations were tested for variation in accumulating ability, and substantial variation was found between populations, particularly at low external zinc concentrations. This variation was unrelated to the contamination status of the soil in which the population grew. In experiment 3, a detailed analysis of three populations showed that between plant variation in zinc accumulation was heritable, with heritabilities between 0.25 and 0.5, and that a small but significant correlation could be found between the field zinc concentration of a maternal plant and the accumulating phenotype of her progeny under standard conditions.
These results indicate that genetic variation in zinc accumulation ability exists in A. halleri, and that it most easily manifested at low external zinc concentrations. At higher concentrations, all plants accumulate substantial amounts of zinc, and the variation between plants with different phenotypes is more likely to be due to chance, local environmental effects, or ontological variation than genes. For instance, differences in root : shoot biomass might lead to differences in the overall rate of accumulation, and, if this character is not genetically controlled, would lead to an environmental effect on leaf zinc concentration. Thus, if a breeder wished to select for increased accumulation ability, it should be possible to obtain a substantial response to selection, but it will be easier to select at low external concentrations, and it is likely that a greater response will be seen in plants grown at low external concentrations. However, the strong correlations between accumulating phenotypes at different external concentrations indicate that plants showing a high concentration at low zinc will show an above average phenotype at high external concentrations as well, and so plants selected at such low concentrations should be more effective accumulators at all concentrations. The results in A. halleri are in contrast to those obtained by Pollard & Baker (1996) who found rather little genetic variation, overall, in Thlaspi caerulescens. However, this species is a self-fertilising species, in contrast to A. halleri, and thus one would expect to find less within populations genetic variance.
There is no relation between accumulating ability and degree of contamination of the site of origin of the seed. Thus the results obtained by Bert et al. (2000), in which they found that a population from an uncontaminated site had a higher intrinsic accumulating ability than one from an uncontaminated site, may simply have been due to the chance selection of the particular populations studied. It is of course possible that the populations sampled from uncontaminated sites were transient, and had been recently colonised from more contaminated areas. However, there is no relation even if only the contaminated sites (most of which supported very large populations of this species) are considered. Note that there is no relation, either, between the concentration of zinc in the maternal plants from highly contaminated sites and the total amount of zinc in the soil around their roots. There is however, a detectable effect of genotype on the zinc concentration of plants collected in the field.
So can we answer the question posed in the introduction: what causes the variation in zinc concentration of specimens of A. halleri in nature? I suggested that either soil metal concentration or plant genotype (or both) could have an effect. At high metal concentrations, it is apparent that external metal concentration has rather little effect on the plant metal concentration, but there is detectable genetic variation both within and between populations. Most of the variation, however, is environmental (i.e. random effects, ontological effects and variations in environmental features uncontrolled and unstudied in these experiments). If plants accumulate metal till they reach a plateau (Fig. 1; Zhao et al., 2000) then a lack of a relationship between external and internal metal concentration might be expected. Genetic variation in concentration at high external concentrations indicates that different genotypes achieve different plateaus.
Thus in contaminated soils, the answer is predominantly environmental effects unrelated to metal contamination, with some evidence of a genetic component. At low metal concentrations, however, the effect of genetic variation is much larger, and it is possible to find individuals that have metal concentrations typical of nonaccumulating species, as well as individuals with much higher concentrations. Thus in these environments, low external concentration can lead to low internal concentration, and a plant with a low zinc concentration could be said to have been affected by both external metal and genotype.