Does zinc protect the zinc hyperaccumulator Arabidopsis halleri from herbivory by snails?
2003; Wiley; Volume: 159; Issue: 2 Linguagem: Inglês
10.1046/j.1469-8137.2003.00783.x
ISSN1469-8137
AutoresSimone B. Huitson, Mark R. Macnair,
Tópico(s)Plant Micronutrient Interactions and Effects
ResumoNew PhytologistVolume 159, Issue 2 p. 453-459 Free Access Does zinc protect the zinc hyperaccumulator Arabidopsis halleri from herbivory by snails? Simone B. Huitson, Simone B. Huitson School of Biological Sciences, University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter EX4 4PS, UKSearch for more papers by this authorMark R. Macnair, Corresponding Author Mark R. Macnair School of Biological Sciences, University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter EX4 4PS, UKAuthor for correspondence: Mark Macnair Tel: +44 1392 263791 Fax: +44 1392 263700 Email: M.R.Macnair@ex.ac.ukSearch for more papers by this author Simone B. Huitson, Simone B. Huitson School of Biological Sciences, University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter EX4 4PS, UKSearch for more papers by this authorMark R. Macnair, Corresponding Author Mark R. Macnair School of Biological Sciences, University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter EX4 4PS, UKAuthor for correspondence: Mark Macnair Tel: +44 1392 263791 Fax: +44 1392 263700 Email: M.R.Macnair@ex.ac.ukSearch for more papers by this author First published: 29 May 2003 https://doi.org/10.1046/j.1469-8137.2003.00783.xCitations: 41AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary • Hyperaccumulation of metals has been proposed to be a defence against herbivores. Here we investigated whether snails discriminated between plants of Arabidopsis halleri, and the F2 of the cross between A. halleri and A. petraea, on the basis of their internal Zn concentration • A. halleri and F2 plants were grown in four different Zn concentrations. Snails preferred F2 plants to A. halleri plants, and preferred plants that had been grown under low external Zn concentrations, but there was no evidence that they discriminated on the basis of internal Zn concentration • F2 plants were germinated on Zn contaminated soil and snails were allowed to eat the seedlings for a range of different time periods. The survivors were grown on, and tested for, Zn accumulation under standard conditions. No difference was found between the different time periods, indicating that the snails had eaten seedlings randomly • The results do not support a hypothesis that high internal Zn concentration protects seedlings from predation or herbivory by snails Introduction Heavy metals are toxic to most plants when present in greater than trace amounts. Thus the discovery of a class of plants that contains percent quantities of normally toxic metals in their leaves (hyperaccumulators: Brooks et al., 1977, see Brooks (1998) and Macnair (2003) for reviews) poses a significant challenge to biology: how and why do they do it? The last decade has seen an explosion of research on these topics (Macnair, 2003). Boyd & Martens (1992), in a classic review, discuss a variety of hypotheses for the adaptive role of hyperaccumulation. They identify five principal hypotheses: increased tolerance; protection against herbivores or pathogens; inadvertant uptake; drought tolerance; allelopathy. In the case of Zn, (Macnair & Smirnoff, 1999) showed that the characters tolerance and accumulation were genetically independent, and there is strong evidence in Thlaspi caerulescens that the hyperaccumulation of Zn does not increase tolerance to the metal, since populations with similar accumulation but very different level of tolerance can be found (Ingrouille & Smirnoff, 1986; Meerts & van Isacker, 1997; Assunção et al., 2001). Thus the first expla-nation is unlikely to be true in the case of Zn (though it may be for Ni, e.g. Krämer et al., 1996, 1997; Boyd et al., 2000). The hypothesis that has attracted the most attention is the defence hypothesis. Plant defence strategies against herbivores can be placed into one of two broad categories, morphological (e.g. trichomes, Walter, 1996 or toughness, Rathcke, 1985) or chemical. Most chemical defences are due to plant secondary compounds (e.g. glucosinolates and cyanogenic glucosides, Bennett & Wallsgrove, 1994), but a number of Australian plant species actively concentrate fluorine in their tissues, which is then used to synthesise the toxic secondary compound fluoroacetate (Twigg & King, 1991). The defence hypothesis suggests that plants similarly extract metals from the soils, and accumulate them in their leaves where their toxic properties can be exploited in order to reduce herbivory or pathogen attack. A number of experiments have compared the defence of plants containing metal with those without. The basic procedure is either to vary the growth medium to achieve variation in metal concentration (i.e. grow plants of one accumulating species either on metal contaminated medium or not) or to compare the accumulator grown in the presence of metal with a related nonaccumulator. Some studies have investigated whether the plants differ in their toxicity to their herbivores, actual or potential; others have investigated whether herbivores’ feeding behaviour is altered when faced with a plant containing high metal levels. Boyd and coworkers (Boyd & Martens, 1994; Martens & Boyd, 1994; Boyd & Moar, 1999; Boyd et al., 2002) have shown that Ni hyperaccumulation makes these plants highly toxic to a number of generalist herbivores. Whether comparing hyperaccumulators grown on normal soil with those grown on Ni, or hyperaccumulators with nonaccumulators, the plants with high Ni concentration are always more toxic to the animals than plants with more normal Ni levels. Thus Boyd & Moar (1999) compared the survival and growth of the polyphagus noctuid moth Spodoptera exigua fed on three species of Strepanthus, only one of which was a hyperaccumulator, which had been grown on either a high Ni or low Ni soil. Survivorship after 10 d was very low on the hyperaccumulator grown on Ni soil. In the nonaccumulator species, there were no differences in survivorship between the two leaf types, but the larvae grown on plants of one of the species (S. tortuosus) that had been grown on high Ni medium, did pupate slower and were smaller than those grown on control leaves. Boyd & Moar (1999) suggest that these sublethal effects may not only be due to the Ni concentration of the leaves, and speculate that the treatment (i.e. plant growth medium) may have affected the plants in other ways than simply in the amount of Ni in their leaves. While the demonstration that the metal in a leaf is toxic implies that a plant will achieve greater protection from herbivores, in practice the plant is still being damaged by a herbivore until the herbivore has ingested the lethal dose. If the plant is small, it might be severely damaged before the herbivore has been killed. It would be more effective as a protectant if herbivores were deterred from eating the leaves. In an elegant experiment, Pollard & Baker (1997) tested this aspect of the interaction. They grew two accessions of T. caerulescens in either a low or high Zn medium. The high Zn plants accumulated 10 × as much Zn in their leaves as the ones grown in the low Zn medium. The plants were presented to three herbivores: locusts (Schistocerca gregaria), slugs (Deroceras caruanae) and caterpillars (Pieris brassicae). All three herbivores showed a preference for the low Zn plants, with the caterpillars rejecting the high Zn leaves without even apparently tasting them. Boyd et al. (2002) have also shown that snails preferred low Ni Senecio coronatus when offered a choice between leaves containing either high or low concentrations of Ni. This effect on the behaviour of herbivores clearly increases the effectiveness of the plants’ defence. There are, however, a couple of criticisms of these experiments that can be made, especially if we are considering the evolution of the trait de novo. One is that the treatment (or geographical source of population in the case of Boyd et al., 2002) is confounded with difference in metal concentration. To achieve the low and high metal groups, the two groups of plants are treated with low and high external levels of the metal involved in hyperaccumulation, the effect of which most likely extends beyond just that of the internal level of that heavy metal. As the treatment is intrinsically linked to the plant's internal heavy metal concentration, the influence of internal heavy metal concentration on herbivory cannot be analysed independently from treatment. Only Jhee et al. (1999) have overcome this objection by using two populations of T. caerulescens that differed in their accumulating phenotype; they also found a protecting effect of Zn. The second problem is that the herbivore is given a choice between two extreme levels of leaf metal concentration. It is more likely that evolution of the hyperaccumulating character was a more stepwise process (Boyd & Moar, 1999), with the actual metal concentration of the plant not as high as that which is seen in hyperaccumulators today, especially if hyperaccumulation evolved on noncontaminated sites, as has been suggested (Macnair et al., 1999). The selection pressure from a herbivore on the accumulating plant must be significant when presented with a choice of leaf tissue with much smaller differences in metal concentration if the defence hypothesis is to be a plausible mechanism for the evolution of this character. This paper uses snails (Helix aspersa) as model generalist herbivores feeding on Arabidopsis halleri plants and F2 individuals from a cross between it and its sister species, A. petraea. The use of the F2 enables us to produce variation in internal Zn concentration even without the use of different growth media, though variation is increased still further by this method. Clearly snails may not be representative of all possible herbivores, but they are a ubiquitous member of the pasture habitats in which A. halleri occurs in nature, and have been used extensively in antifeedant experiments by many authors (e.g. Compton & Jones, 1985; Boyd et al., 2002). Materials and Methods Provenance of plant material Arabidopsis halleri (L.) O’Kane & Al-Shehbaz plant material was originally collected from a population near Langelsheim, West Germany (Macnair et al., 1999). This population is the Innerste population of Macnair (2002). Plants of this population were polycrossed amongst each other to produce the seeds used in the experiments described here. Details of polycrossing are given in Macnair (2002). Three A. halleri individuals were crossed to individuals of A. petraea (= A. lyrata ssp. petraea (L.) O’Kane & Al-Shehbaz) from a population at Unhošt’ in Bohemia (Macnair et al., 1999). Fifteen F1 plants with various parents were polycrossed together to give a bulk F2, which segregates for both tolerance and accumulation (Macnair et al., 1999). Plants of A. petraea were also polycrossed to produce the seed used in these experiments. Plant Zn determination Leaf Zn concentration was determined on small leaf fragments or whole seedlings using the colourimetric method of Macnair & Smirnoff (1999). This method gives plant Zn concentrations in fresh weight terms. In order to allow easy conversion of fresh weights concentrations to dry weight concentrations (which are more generally used in the literature) 17 plants of varying genotype and growth medium were weighed fresh and then dried for 3 d at 70°C until constant weight. The d. wt : f. wt ratio was 20.3 ± 1.0%, and thus the classic hyperaccumulator threshold of 10 000 p.p.m. corresponds to approximately 30 µmol g f. wt−1. Plant cultivation All experiments described here were performed in a growth room with a 10-h day and temperature 25°C (day) and 15°C (night). Except where otherwise stated, plants were germinated on sand, and transferred to trays containing the hydroponic solution described elsewhere (Macnair & Smirnoff, 1999; Macnair, 2002) in which iron is as FeEDDHA. Zn was added at concentrations noted in different experiments as ZnSO4. Solutions were changed weekly and aerated continuously. Statistics Data were analysed using SPSS v10.1. Analyses of Variance or Covariance were conducted using the General Linear Model (GLM) method. The approximate normality of the residuals and assumptions of anova were checked using standard visual tests (plot of residual against predicted values; G-G plot). Experiment 1: characterisation of the F2 In order to characterise the variation in the F2, c. 500 F2 were grown for 5 wk in a solution containing 10 µm Zn. They were then transferred to a solution containing 100 µm Zn for 4 wk. The Zn concentration was determined on two different leaf fragments, and the values averaged. Experiment 2: herbivory on mature plants In this experiment individual adult snails were presented with six F2 plants and two A. halleri plants with varying concentrations of Zn in their leaves. In order to generate substantial variation in leaf Zn concentration, A. halleri and F2 plants were grown in 10-l trays amended with 10, 50, 100 or 250 µm Zn. A. halleri and the F2 seedlings were randomly assigned to positions within these growth trays. The trays were kept under controlled conditions in a growth room until the plants were large enough to be used in feeding studies (rosettes at least 5 cm diameter). The leaf Zn concentration of each plant was then determined, and the F2 plants were ranked by this variable. The plants were then split into six groups by plant rank. For each experimental tray, one F2 plant was randomly taken from each group, along with two randomly chosen A. halleri plants. For each plant, the roots were gently blotted and the whole plant was weighed, and then placed randomly in the feeding tray. The feeding trays were 1-l trays containing the standard growth solution amended with 50 µm Zn as ZnSO4. Pieces of polystyrene were cut to the same size as the trays and eight holes cut into which the plants were inserted. Fifteen feeding trays were set up, each with six F2 plants and two A. halleri. Adult snails (Helix aspersa) of approximately equal size, which had been collected from around the glasshouses of the University of Exeter, were added to 12 of the trays (one snail per tray) and the remaining three were used as controls. After 4 d the plants were removed from the trays, blotted and reweighed. The difference in weight was taken as a measure of the degree of herbivory on the plant; note that positive values indicate net growth of the plants over the period of the experiment. Experiment 3: pattern of Zn uptake by small seedlings Six plastic boxes were lined with eight layers of absorbent tissue wetted with 30 ml standard nutrient solution amended with either 10 (two boxes), 100 (two boxes), 500 (one box) or 1000 µM (one box) Zn. The mass of each box was recorded and 150 A. halleri or A. petraea seeds were sown in each box, with A. halleri sown in all Zn concentrations but A. petraea sown only in 10 or 100 µm Zn. Seeds were checked for germination on a daily basis and removed to grow in hydroponic culture in nutrient solution with the same Zn concentration as in the germination box. The mass of the box was adjusted daily to the same mass as recorded at the beginning of the experiment with distilled water to compensate for evaporation. Absorption of minerals by the seeds from the nutrient solution was assumed to be negligible. The first measurements of leaf Zn concentration were made on seedlings that were 2 old and at daily increments, up to 15 d old. The age at which an individual seedling was assayed was determined at random. As the leaf mass of a single young seedling was too low to allow an accurate assay of Zn concentration, leaves from several seedlings of the same age were pooled for each sample. For analysis of the data, the age of a seedling was measured from germination to when the sample was recorded. Experiment 4: herbivory by snails on small seedlings This experiment was designed to test whether snails could discriminate between very small seedlings according to their accumulating phenotype. F2 seeds were sown in potting compost and allowed to germinate (day 0). In order to increase the amount of Zn in seedlings, the soil was supplemented with 5000 p.p.m. Zn (equivalent to that measured in contaminated soils by Bert et al., 2000) in the form of ZnO. On day 12, the trays were soaked with nutrient solution containing 5000 µm Zn, which was raised to 0.17 m Zn on day 13. The feeding trials were begun on day 15. Nine trays of seedlings were used, with one snail (Helix aspersa) added to each of seven trays and the seedlings in the remaining two trays used as controls with no snails added. Eight hours after addition of the snails (still day 15), the snails in two of the trays were removed. In a similar manner, snails were removed from two remaining trays 22.5 h after the beginning of the feeding trail (day 16) and the three last snails were removed after 47.5 h (day 17). On day 19, in order to assess the ability of each F2 seedling to hyperaccumulate Zn, all the surviving seedlings from each of the trays were transferred to hydroponic solution, supplemented with 100 µm Zn. The plants were allowed to grow for 2 wk, after which they were assayed for leaf Zn concentration. If the snails are discriminating between seedlings, there should be a difference between the mean Zn accumulating phenotype of survivors after predation and those not exposed to predation. Results Experiment 1 Figure 1 gives the distribution of Zn accumulation in the F2. It is clear that this F2 has a more-or-less normal distribution of accumulating phenotypes. This sample was also tested at 10 µm and higher concentrations of Zn, and the normal distribution was maintained (data not shown). Figure 1Open in figure viewerPowerPoint Histogram of the leaf Zn concentration of 513 F2 plants from the cross Arabidopsis halleri × Arabidopsis petraea. The plants had been grown in hydroponic culture, for 5 wk at 10 µm, followed by 4 wk at 100 µm. Experiment 2 Overall, A. halleri was able to accumulate more Zn than the F2 plants (means: A. halleri= 15.26 µmoles g f. wt−1, F2 = 7.18 µmoles g f. wt−1, t-test: t = 6.58, d.f. = 118, P < 0.001). Within the F2, the plants grown in higher external Zn accumulated more than those grown in lower external Zn (anova: F3,86 = 32.51, P < 0.001). However, the mean leaf Zn concentration of the plants in the feeding trays did not differ significantly (anova: F14,75 = 0.04, P = 0.963), hence the snails were subjected to similar levels of Zn in their diet. The change in mass of the plants in the experimental feeding trays also did not differ significantly between trays (anova: F11,84 = 0.57, P = 0.847) showing that the snails were feeding at similar rates in each of the feeding trays. The snails ate the F2 in preference to A. halleri (Fig. 2). The mean change in mass for the two plant types was found to be significantly different (Means: A. halleri = 0.924 g, F2 = −0.389 g), showing that A. halleri increased in mass but the F2 decreased in mass due to herbivory (t-test for samples with unequal variances: t = 4.23, d.f. = 28.8, P < 0.001). Figure 2Open in figure viewerPowerPoint The relationship between the amount of a plant eaten by a snail, and the Zn concentration of the plant. Individuals from an F2 cross between Arabidopsis halleri and Arabidopsis petraea, together with A. halleri plants, were grown in one of four Zn concentrations. Plant Zn level was determined, and six F2s and two A. halleri plants were chosen with a range of internal Zn concentrations and grown with a single snail for 4 d. Plant f. wt was determined before and afterwards; the change in mass is plotted. Positive values represent net growth over the experiment. Solid circles: A. halleri plants; open squares: F2 plants grown at 10 µm Zn; open triangles: F2 plants grown at 50 µm Zn; open diamonds: F2 plants grown at 100 µm Zn; open circles: F2 plants grown at 250 µm Zn. Figure 2 shows the relationship between the amount of each plant eaten and the leaf Zn concentration. It is clear that in the F2 plants there is an overall negative correlation between herbivory and Zn concentration (F2 plants: r = 0.234, d.f. = 71, P < 0.05). However, when these data were analysed by GLM with original growth medium as a fixed factor, and leaf Zn concentration as a covariate, there is no significant relationship between change in mass and leaf Zn concentration (F1,67 = 2.16, P = 0.147) but there is a significant effect of growth medium (F3,67 = 6.94, P < 0.001). If the interaction is included in the model, both the growth medium and interaction terms are highly significant (medium: F3,64 = 16.86, P < 0.001; interaction: F3,64 = 8.89, P < 0.001) and the leaf Zn concentration term becomes marginally significant (F1,64 = 5.18, P = 0.026). However, the overall slope of the covariate is negative, that is suggesting that increased internal Zn concentration results in increased herbivory. The control trays were analysed to determine whether leaf Zn concentration or growth medium had an effect on change in mass without the influence of snails. No significant correlation between change in plant mass and leaf Zn concentration was found for the control F2 data overall (r = −0.186, d.f. = 17, P = 0.460), nor was there any detectable effect of growth medium on plant growth rate (GLM: F3,13 = 0.48, P = 0.704). Thus the differences in pattern detected in the herbivory treatment can be attributed to the feeding behaviour of the snails. Experiment 3 The A. halleri seeds germinated slightly faster than the A. petraea seeds, and the A. halleri seeds in high Zn concentrations germinated faster than those in lower Zn concentrations (data not shown). However, the time taken to germinate did not affect their subsequent Zn concentration (F13,8.7 = 2.1, P = 0.139). The leaf Zn concentration of the seedlings by age is shown in Fig. 3. Only the data from A. halleri seedlings is shown since A. petraea seedlings did not accumulate significant amounts of Zn over this time period. The youngest seedlings have a low Zn concentration, which increases with age (age of seedling term in anova: F1,142 = 122.5, P < 0.001) and this increase is higher in the seedlings that have been germinated and grown in higher external Zn concentrations (external Zn concentration term: F3,94.6 = 128.8, P < 0.001). Note that in the high external Zn concentrations, these seedlings attain leaf Zn concentrations of over 30 µmol g f. wt−1, that is over 1% by dry weight, showing that the hyperaccumulation phenotype can be attained quickly (though not immediately). In lower concentrations, however, the seedlings plateau out at a much lower leaf Zn concentration. Figure 3Open in figure viewerPowerPoint The accumulation of Zn by seedlings of Arabidopsis halleri in various external concentrations of Zn. At day 0, newly germinated seedlings were transferred to nutrient solutions with the requisite concentration of Zn. Circles: 10 µm; triangles: 100 µm; squares: 500 µm; diamonds: 1000 µm. Experiment 4 The distribution of leaf Zn concentration after each herbivory treatment followed by 14 d in 100 µm Zn amended nutrient solution is shown in Fig. 4. If the herbivores had chosen to eat seedlings depending on their internal Zn concentration, then there should have been a change in the mean Zn accumulation of seedlings depending on how long they had been exposed to predation. However, there is no significant difference between the means of these treatments (anova: F3,211 = 0.972, P = 0.407), indicating that the herbivores were not able to select on the basis of the seedling's ability to accumulate Zn. Figure 4Open in figure viewerPowerPoint Histogram showing the Zn accumulating phenotype of surviving F2 seedlings that had been exposed to predation for varying times by snails. Seeds were germinated in Zn contaminated soil and exposed to snails after 15 d. After the snails had been removed, the survivors were grown on in 100 µm Zn for 2 wk. The histograms show the proportion of the survivors in each accumulating phenotype. The numbers of survivors in each class were: 0 h: 72; 8 h: 82; 22.5 h: 17; 47.5 h: 43. Discussion In these experiments, we have used both genetic segregation and environmental differences to generate variation between plants in their internal Zn concentration. We found that snails avoid A. halleri plants, and discriminate between F2 plants on the basis of the environment in which they were grown, but we did not find any evidence that snails discriminated between F2 plants on the basis of their internal Zn concentration when they had been grown under the same conditions. There has been only other study where genetic variation was used to generate the variation in Zn concentration. Jhee et al. (1999) used two different populations of T. caerulescens that differed in their Zn uptake phenotype, and found that the plants with less Zn were attacked more than those with higher Zn concentrations. However, the differences between the populations were quite large, and the populations may have differed in other ways than just Zn concentration. If Zn hyperaccumulation evolved in a low Zn environment, as seems probable, then in the initial stages variation in Zn concentration between plants will have been low and continuous, and thus the F2 variation used in this study may be a more realistic model of the situation than the use of populations, which differ substantially in Zn concentration. In experiment 2, we found that the major cause of the differences in herbivory rates was not the internal Zn concentration, but the medium in which the plants had been grown (Fig. 2). We are unable to speculate on what has made the plants less palatable, but it could be that the different treatments induce differences in other plant defensive compounds (e.g. glucosinolates) or even the toughness of the leaves. If the effects observed here are general, then caution must be exercised in interpreting previous experiments testing the defence hypothesis since they generally use either differences in growth medium or differences in species to generate the variation in plant metal concentration. These experiments do not provide support for the defence hypothesis in the case of Zn. Snails did not discriminate between large plants on the basis of their internal Zn concentration (experiment 2). In experiment 4, snails were presented with small F2 seedlings, which will have differed in Zn concentration through genetic segregation. Plants are most vulnerable to herbivory while they are seedlings: once they have attained reasonable size, they are unlikely to be killed outright by herbivory, at least by invertebrate herbivores such as snails. Experiment 3 showed that about 14 d after germination A. halleri seedlings had attained substantial internal Zn concentrations when grown in contaminated environments. Assuming that accumulating F2 plants will show the same pattern of accumulation, we can presume that the F2 seedlings had a range of internal Zn concentrations at the point at which they were exposed to snails. In experiment 4, we exposed batches of seedlings to herbivory for a varying length of time. If snails preferred seedlings with low Zn concentration, then they should have preferentially eaten seedlings with low-accumulation genotypes and the survivors should have shown a progressively increasing accumulation phenotype. They did not. It is important to note that the selective forces currently acting on a character may not be the same as those responsible for its initial evolution. Even if herbivores are deterred by the high levels of metal present in hyperaccumulators growing on contaminated sites, it does not follow that the same mechanism must have been responsible for its initial evolution. Unless hyperaccumulation is governed by major genes (and there is no evidence for this, Macnair et al., 1999, and see Fig. 1), selection will presumably have had to act to create the current levels of accumulation by selecting on a continuous distribution. It is probable that Zn hyperaccumulation evolved in an uncontaminated environment, since all of A. halleri's closest relatives cannot grow on such soils, and in central Europe A. halleri is found as a normal component of the flora of uncontaminated sites (Clapham & Akeroyd, 1993; Bert et al., 2000, 2002). It is parsimonious to presume that A. halleri only colonised Zn contaminated soils after such soils had become more widespread as a result of human activity. Experiment 3 showed that young seedlings growing in an uncontaminated environment (simulated in this experiment by an external Zn concentration of 10 µm) accumulate very little Zn while they are small. Thus at this most vulnerable stage of a plant's life history, A. halleri does not have sufficient Zn in its leaves to provide any protection. Even when grown at 100 µm (a concentration toxic to A. thaliana and A. petraea) the leaf Zn concentration is well below the levels shown by others (Pollard & Baker, 1997) to be protective. Experiment 2 did not give any indication that larger plants were more protected by Zn than seedlings. Thus these experiments have not provided support for the defence hypothesis for Zn hyperaccumulation in A. halleri, either as a process maintaining hyperaccumulation or as a mechanism for its initial evolution. Of the other hypotheses advanced by Boyd & Martens (1992), increased tolerance has been discounted by the results of Macnair et al. (1999). Allelopathy and drought tolerance are also unlikely (Macnair, 2003). In the absence of a plausible adaptive hypothesis, the final suggestion of Boyd & Martens (1992), inadvertant uptake, must be considered carefully. Davis et al. (2001) have also argued in favour of this hypothesis: they found no evidence that the Ni hyperaccumulator Psychotria douarrei had reduced organic defences, and that a variety of other metals were at a higher than normal concentration in this species. The inadvertant uptake hypothesis deserves explicit test. Acknowledgements Simone Huitson was supported by a NERC studentship, receipt of which is gratefully acknowledged. References Assunção AGL, Da Costa Martins P, De Folter S, Vooijs R, Schat H, Aarts MGM. 2001. Elevated expression of metal transporter genes in three accessions of the metal hyperaccumulator Thlaspi caerulescens. Plant, Cell & Environment 24: 217– 226. Wiley Online LibraryCASWeb of Science®Google Scholar Bennett RN, Wallsgrove RM. 1994. Secondary metabolites in plant defence mechanisms. New Phytologist 127: 617– 633. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Bert V, Bonnin I, Saumitou-Laprade P, De Laguérie P, Petit D. 2002. Do Arabidopsis halleri from nonmetallicolous populations accumulate zinc and cadmium more effectively than those from metallicolous populations? New Phytologist 155: 47– 57. Wiley Online LibraryCASWeb of Science®Google Scholar Bert V, Macnair MR, De Languerie P, Saumitou-Laprade P, Petit D. 2000. Zinc tolerance and accumulation in metallicolous and nonmetallicolous populations of Arabidopsis halleri (Brassicaceae). New Phytologist 146: 225– 233. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Boyd RS, Davis MA, Wall MA, Balkwill K. 2002. Nickel defends the South African hyperaccumulator Senecio coronatus (Asteraceae) against Helix aspersa (Mollusca: Pulmonidae). Chemoecology 12: 91– 97. CrossrefCASWeb of Science®Google Scholar Boyd RS, Martens SN. 1992. The raison d’être for metal hyperaccumulation by plants. In: AJM Baker, J Proctor, RD Reeves, eds. The vegetation of ultramafic (serpentine) soils. Andover, UK: Intercept, 279– 289. Web of Science®Google Scholar Boyd RS, Martens SN. 1994. Nickel hyperaccumulated by Thlaspi montanum var montanum is acutely toxic to an insect herbivore. Oikos 70: 21– 25. CrossrefCASWeb of Science®Google Scholar Boyd RS, Moar WJ. 1999. The defensive function of Ni in plants: response of the polyphagous herbivore Spodoptera exigua (Lepidoptera: Noctuidae) to hyperaccumulator and accumulator species of Streptanthus. Oecologia 118: 218– 224. CrossrefPubMedWeb of Science®Google Scholar Boyd RS, Wall MA, Watkins JE. 2000. Correspondence between Ni tolerance and hyperaccumulation in Streptanthus (Brassicaceae). Madroño 47: 97– 105. Google Scholar Brooks RR. 1998. Plants that hyperaccumulate heavy metals. Wallingford, UK: CAB International. Google Scholar Brooks RR, Lee J, Reeves RD, Jaffré T. 1977. Detection of metalliferous rocks by analysis of herbarium specimens of indicator plants. Journal of Geochemical Exploration 7: 49– 77. CrossrefCASWeb of Science®Google Scholar Clapham AR, Akeroyd JR. 1993. Cardaminopsis. In: TG Tutin et al. , eds. Flora Europaea 1, 2nd edn . Cambridge, UK: Cambridge University Press. Google Scholar Compton SG, Jones DA. 1985. An investigation of the responses of generalist herbivores to cyanogenesis in Lotus corniculatus L. Biological Journal of the Linnean Society 26: 21– 38. Wiley Online LibraryWeb of Science®Google Scholar Davis MA, Pritchard SG, Boyd RS, Prior SA. 2001. Developmental and induced responses of nickel-based and organic defences of the nickel-hyperaccumulating shrub, Psychotria douarrei. New Phytologist 150: 49– 58. Wiley Online LibraryCASWeb of Science®Google Scholar Ingrouille MJ, Smirnoff N. 1986. Thlaspi caerulescens J. & C. Presl. (T. alpestre L.) in Britain. New Phytologist 102: 219– 233. Wiley Online LibraryWeb of Science®Google Scholar Jhee EM, Dandridge KL, Christy AM, Pollard AJ. 1999. Selective herbivory on low-zinc phenotypes of the hyperaccumulator Thlaspi caerulescens (Brassicaceae). Chemoecology 9: 93– 95. CrossrefCASWeb of Science®Google Scholar Krämer U, Cotter-Howells JD, Charnock JM, Baker AJM, Smith JAC. 1996. Free histidine as a metal chelator in plants that accumulate nickel. Nature 379: 635– 638. CrossrefCASWeb of Science®Google Scholar Krämer U, Smith RD, Wenzel WW, Raskin I, Salt DE. 1997. The role of metal transport and tolerance in nickel hyperaccumulation by Thlaspi goesingense Hálácsy. Plant Physiology 115: 1641– 1650.CrossrefCASPubMedWeb of Science®Google Scholar Macnair MR. 2002. Within and between population genetic variance for zinc accumulation in Arabidopsis halleri. New Phytologist 155: 59– 66. Wiley Online LibraryCASWeb of Science®Google Scholar Macnair MR. 2003. The Hyperaccumulation of metals by plants. Advances in Plant Science (in press.) Google Scholar Macnair MR, Bert V, Huitson SB, Saumitou-Laprade P, Petit D. 1999. Zinc tolerance and hyperaccumulation are genetically independent characters. Proceedings of the Royal Society of London, Series B. 266: 2175– 2179. CrossrefCASPubMedWeb of Science®Google Scholar Macnair MR, Smirnoff N. 1999. Use of zincon to study uptake and accumulation of zinc by zinc tolerant and hyperaccumulating plants. Communications in Soil Science and Plant Analysis 30: 1127– 1136. CrossrefCASWeb of Science®Google Scholar Martens SN, Boyd RS. 1994. The ecological significance of nickel hyperaccumulation – a plant chemical defense. Oecologia 98: 379– 384. CrossrefPubMedWeb of Science®Google Scholar Meerts P, Van Isacker N. 1997. Heavy metal tolerance and accumulation in metallicolous and non-metallicolous populations of Thlaspi caerulescens from continental Europe. Plant Ecology 133: 221– 231. CrossrefWeb of Science®Google Scholar Pollard AJ, Baker AJM. 1997. Deterrence of herbivory by zinc hyperaccumulation in Thlaspi caerulescens (Brassicaceae). New Phytologist 135: 655– 658. Wiley Online LibraryCASWeb of Science®Google Scholar Rathcke B. 1985. Slugs as generalist herbivores-tests of three hypotheses on plant choices. Ecology 66: 828– 836. Wiley Online LibraryWeb of Science®Google Scholar Twigg LE, King DR. 1991. The impact of fluoroacetate-bearing vegetation on native Australian fauna – a review. Oikos 61: 412– 430. CrossrefCASWeb of Science®Google Scholar Walter DE. 1996. Living on leaves: mites, tomenta, and leaf domatia. Annual Review of Entomology 41: 101– 114. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume159, Issue2August 2003Pages 453-459 This article also appears in:Heavy metals and plants FiguresReferencesRelatedInformation
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