Physiological responses of plants to heavy metals

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Chemical Speciation and Bioavailability 1 ( 1 ) , 7-17, March 1989

REVIEW

Physiological responses of plants to heavy metals and the quantification of tolerance and toxicity A.J.M. Baker* and P.L Walker Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK

Abstract Techniques available for assessing the tolerance of plants to heavy metal toxins are reviewed. All are based on physiological responses and range from long-term growth trials in metal-contaminated substrates, to rapid cytological tests. Problems associated with the ecophysiological interpretation of in vitro measurements of tolerance are considered. The implications of multiple tolerance, co-tolerance, constitutional tolerance, inducible tolerance and possible stimulatory effects of metals on plant responses are discussed. Keywords: Plants, metal tolerance, metal toxicity.

Introduction There has been an ever-increasing awareness over the last two decades of the potency of heavy metals as environmental pollutants. Their persistence in the environment and presence in a variety of inorganic and complexed chemical forms result in their becoming incorporated into biological cycles where they can exert long-term toxicity effects. Heavy metal pollution can bring about severe phytotoxic action; it can also act as a powerful force on plant populations leading to the directional selection of tolerant genotypes. Considerable attention has been focused on these evolutionary processes (for reviews see Antonovics, Bradshaw and Turner, 1971; Baker, 1987) and the

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physiological responses of plants to the metal toxins involved. In all such studies, there is a need to quantify the toxic effects of the metals concerned. A wide range of plant responses have been employed for this purpose and it is the aim of this review to bring together such disparate information. The tests described may find considerably wider usage in other studies of phytotoxicity and bioavailability of toxic elements. The ecophysiological significance of experimental measurements of heavy metal tolerance requires cautious interpretation particularly in the light of anomalous responses. These are also considered in depth in this review. All plants respond to increases in heavy metal concentrations in their immediate environment. The nature,

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Dose (Available Soil Concentration) Figure 1 Generalised yield-dose response curve to illustrate the effects of changes in available metal concentrations in the soil on plant performance. (Reprinted with modification from Berry and Wallace, 1981, p.14, by courtesy of Marcel Dekker, Inc.)

Review: Physiological responses of plants direction and magnitude of these responses will depend on the sensitivity of the individual, the intensity (concentration and duration) of exposure, the metal concerned and the form in which it is present. From experimental studies it is possible to construct yield-dose response curves, where yield can represent a growth parameter ranging from biomass production in the long-term to estimates of root growth inhibition in the short-term. Figure 1 illustrates such a generalised response curve. It provides a useful basis for the definition of deficiency (in the case of essential trace elements), tolerance and toxicity (Berry and Wallace, 1981). The precise form of the response curve will depend on relative species sensitivities which, in turn, will determine their usefulness in bioassay studies for any particular metal toxin. Differential effects of an appropriate metal concentration, or concentration range, on the performance of different species, populations or genotypes can therefore be used to quantify both sensitivity and tolerance.

are outlined below to illustrate the techniques which have been employed, ranging from more extended comparative studies of whole organisms to short-term screening at the cellular level. Seedling survival Few studies have examined whole organism responses in terms of the superior fitness of tolerant genotypes in their native, contaminated habitat. Walley et al.t (1971) examined the survival of a commercial, non-tolerant strain of the grass Agrostis tenuis Sibth. (= A. capillaris L.) and a copper-tolerant race on a range of copper-contaminated/ normal soil mixtures. They demonstrated significant differences in germination responses, but found that percentage survival was the most useful measure of performance, since many seedlings which had successfully germinated subsequently failed to grow and remained characteristically stunted. Of the presumed non-tolerant seedlings, 5% germinated on the unameliorated mine-spoil treatment but none of these survived for more than four months (Figure 2). Other workers (Allen and Sheppard, 1971; Karataglis, 1980) have also employed differential survivorship as a measure of tolerance. Karataglis (1980) also recorded the percentage of seedlings producing roots as a guard against including those individuals which germinated but failed to root. Cox (1979) found that germination of non-tolerant Deschampsia cespitosa (L.) Beauv. seed was similar on control and copper/nickel- contaminated soils and that differences were only apparent in the numbers surviving. As expected, tolerant seedlings showed superior survival to the non-tolerants on contaminated soil. However, one of the tolerant populations achieved a higher germination on the contaminated soils than the control implying a "need" for elevated levels of copper. Other species studied which have shown differential survivorship in the tolerant race include: Mimulus guttatus DC. (Allen and Sheppard, 1971), Silene cucubalus Wib. (= S. vulgaris (Moench) Garcke) (Schiller, 1974), Agrostis

Techniques for the Quantification of Metal Tolerance Approaches to the study of metal tolerance can be conveniently divided into those employing measures of growth or vigour and investigations of metal uptake and accumulation. Only the former will be considered in detail here as metal uptake studies have recently been reviewed in depth elsewhere (Baker and Walker, 1989). The most clear-cut separation between tolerant and non-tolerant individuals is in their ability to establish, survive and reproduce in metal-contaminated substrates. To assess such differences involves studying the complete life-cycle and employing some measure of fitness. Experimental studies of this nature are fraught with problems and usually involve long-term growth trials. Accordingly, a range of more easily and rapidly measured physiological responses have been used to screen for plant responses to heavy metal contamination. These

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Figure 2 Number of survivors (%)from normal and copper mine populations of Agrostis capillaris L. after 4 months' growth on different mine waste/uncontaminated soil mixtures, ranging from 3:1 to unameliorated waste. (Redrawn from Walley et al., 1971, and reproduced with permission of the Genetical Society of Great Britain.)

A. J. M. Baker & P. L. Walker stolonifera L. (Maschmeyer and Quinn, 1976), Festuca rubra L. (Karataglis, 1980). Investigations such as those mentioned above provide provisional evidence for population differentiation within a species with respect to heavy metal tolerance. The results of tests based on survival must, however, be interpreted cautiously as the superior ability of individuals from one race to survive may also be linked to tolerance of other factors such as low nutrient availability and drought which are often features of mine-spoils (Antonovics et al., 1971). However, Walley et al. (1971) confirmed that the individuals better able to survive in a copper-contaminated soil were indeed tolerant to toxic levels of copper in a solution culture. It is also unusual for only one heavy metal to be elevated in concentration in a contaminated substrate. This reduces the effectiveness of such studies, since comparisons cannot be made between the specific response of several different tolerant populations to any one metal. Biomass Growth performance, either in terms of biomass yield or growth rate, provides a ready means of assessing intraspecific differences in the effects of metal treatment (Verkleij and Bast-Cramer, 1985; Verkleij, Prast and Ernst, 1986; Ingrouille and Smirnoff, 1986). Mathys (1977, 1980) comments on the advantage of total biomass measurements in assessing long-term effects. In one experiment, the biomass of several plant species grown in elevated levels of zinc for four weeks

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was expressed as a percentage of the control. The non-tolerant plants showed decreased growth whilst the tolerant ones were stimulated and seemed to require elevated zinc concentrations for optimal growth. Verkleij et al. (1986) showed differences in tolerance to cadmium in Silene cucubalus races by fresh weight yield (Figure 3). Similarly, Gerakis, Veresoglou and Sakellariadis (1980) demonstrated that cultivars of Beta vulgaris L. differed in their sensitivity to lead, as seen in their total dry weight yields. It is a common observation that the rates of dry matter production and the biomass yield of tolerant plants are lower than their non-tolerant counterparts (Ernst, 1976; Wilson, 1988). This reduction is believed to be a corollary of the energy expenditure in metal tolerance mechanisms (Ernst, 1976). The "costs" of tolerance may thus reduce the fitness of tolerant plants when grown in uncontaminated soils (Cook, Lefebvre and McNeilly, 1972; Cox and Hutchinson, 1981; Bradshaw, 1984). Metal tolerance in algae has also been expressed as differences in biomass although cultures of cells rather than single organisms are studied. Hall (1980) found that wet weight of algal samples, measured after a standard time of vacuum filtration, correlated well with dry weight and also reflected differences in the tolerance of two populations of Ectocarpus siliculosus (Dillw.) Lyngbye to copper, cobalt and zinc. Russell and Morris (1970) demonstrated tolerance to copper in a similar population of this species by estimating percentage change in culture volume after slow centrifugation. Stokes, Hutchinson

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Figure 3 Mean fresh weight of.seedlings (g), (± standard error) of cadmium-tolerant (A) and non-tolerant (•) populations of Silene cucubalus grown in cadmium treatment solutions. (Drawn from data of Verkleij et ai, 1986.)

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Review: Physiological responses of plants

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Height of seedling (mm) Figure 4 The relationship between index of copper tolerance (%) and height of seedlings (mm) for a normal population of Agrostis capillaris after four months' growth on a 6:1 copper mine waste/soil mixture. The individual marked (o) was a seedling from a separate, but parallel experiment. (Redrawn from Walley et al., 1986, and reproduced by kind permission of the Genetical Society of Great Britain.) and Krauter (1973) established that cell counts during exponential growth correlated with dry weight and also reflected population differences in the tolerance of Chlorella and Scenedesmus spp. to copper and nickel. Shoot growth Many of the studies of higher plants cited above (e.g. Walley et al, 1971; Allen and Sheppard, 1971; Maschmeyer and Quinn, 1976; Karataglis, 1980) also employed siioot yield and height as additional measures of performance to differentiate the responses of tolerant and non-tolerant individuals. Walley et al. (1971) found that the height of A. tennis seedlings grown on a slightly diluted mine spoil showed a strong correlation with the degree of copper tolerance as measured in solution culture (Figure 4). Differences in tolerance to cadmium were reflected in the shoot weights of populations of A. tenius and Festuca rubra on soil artificially contaminated with CdO (Hertstein and JSger, 1986). Final shoot weight also discriminated between the response of serpentine and acidic soil clones of F. rubra to nickel and magnesium (Johnston and Proctor, 1981). Root growth Early studies of heavy metal tolerance in higher plants (Bradshaw, 1952; Wilkins, 1957) demonstrated that root growth was particularly sensitive to the presence of metal toxins. Root biomass, total root length, root number and rates of root elongation have all been employed to measure tolerance. Thus, although revealing differences in sensitivity of Beta vulgaris cultivars by total dry weight yield, Gerakis et al. (1980) found that the greater sensitivity of one cultivar to field applications of lead, was shown more clearly by root biomass. Significant differences between the response of tolerant and non-tolerant populations of the grass Holcus lanatus L. to cadmium were shown by root weight but more so by root extension

measurements (Brown, 1983). Many of the experiments designed to demonstrate metal tolerance in plants are based on the original techniques developed by Wilkins (1957, 1978) and Jowett (1964). In essence, solution culture is used to study the root growth responses of individual plants or clones, to an elevated level of the metal as measured by rate of root elongation. A comparison of performance in the presence of metal (treatment solution) with that in its absence (control solution) is used to calculate an index of tolerance. There are now many variants of this basic technique which has proved a satisfactory and rapid method for measuring metal tolerance. Figure 5 shows the typical responses of Cd-tolerant and non-tolerant genotypes in this type of test The methodology and limitations of the technique have been fully reviewed elsewhere (Wilkins, 1978; Humphreys and Nicholls, 1984; Baker, 1987). In general, the rapidity and simplicity of the method seem to far outweigh its limitations. The root elongation test has been used to quantify metal tolerance in a wide range of angiosperms (Antonovics et al., 1971; Antonovics, 1975). Although primarily designed for higher plants, Briggs (1972) has successfully employed a similar technique to measure lead tolerance in vegetative propagules of the liverwort Marchantia polymorpha L. Pollen tube growth The root elongation technique has been applied in modified form to measure metal tolerance by means of pollen tube growth in liquid culture. Searcy and Mulcahy (1985a,b), removed pollen from tolerant and non-tolerant Mimulus guttatus, Silene dioica (L.) Clairv. and S.alba (Mill.) Krause and incubated it in media containing a range of copper and zinc concentrations. The tolerance was expressed as differences in percentage pollen germination and pollen tube lengths. These differences reflected those seen in the parents by means of a conventional rooting test.

A. J. M. Baker & P. L. Walker Lepp and Dickinson (1986) used a similar method to demonstrate differences in the copper tolerance of pollen collected from two stands of the same cultivar of Coffea arabica L. Protoplasmic resistance and membrane damage The ability of cells (generally those of epidermal strips removed from leaves) to recover from plasmolysis after incubation in metal treatment solutions, has also been used to measure metal tolerance (Repp, 1963, 1973; Gries, 1966; Ernst, 1972). The tissue strips are exposed to a range of metal concentrations (including a control) and cell vitality is then scored as the percentage of cells plasmolyzed after immersion in molar glucose solution. A resistance limit is often defined as that metal concentration in which « 80% of the sheets of tissue survive. This rapid technique has been used to demonstrate differences in tolerance to heavy metals in both mosses (Url, 1956) and in higher plants to zinc (Gries, 1966, 1968), copper (Repp, 1963) and nickel (Ernst, 1972), The physiological basis of this technique is the differential effect of heavy metal ions in bringing about membrane damage in cells of tolerant and non-tolerant plants. Similarly, measurements of rates of potassium efflux from roots in solution culture after metal treatment, have also been used to investigate the same phenomenon (Wainwright and Woolhouse, 1975). Responses at the cellular and subcellular level Differential responses to heavy metals between tolerant and non-tolerant plants can also be demonstrated at the cellular and subcellular levels. As root growth is a result of cytokinesis, cell extension and differentiation, metal-induced retardation of root growth could be due to toxic influences acting on any

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combination of these processes (Powell, Davies and Francis, 1986). Clarkson (1965) demonstrated a direct effect of aluminium on mitosis in roots ofAllium cepa L. but did not test plants which differed in sensitivity. Powell et al. (1986) have shown that zinc tolerance in two Festuca rubra cultivars is expressed at both the whole root and cellular levels. The greater sensitivity of a non-tolerant cultivar was in part due to a lower zinc concentration threshold for inhibition of cell division and also a greater effect of zinc on the duration of the Gl phase of the cell cycle. Wainwright and Woolhouse (1975) found that differences in metal tolerance in Agrostis capillaris races were also manifested as effects on cell elongation, with a greater inhibition occurring in the sensitive ecotype. Tissue culture has been used to show differences in metal tolerance for higher plants. Wu and Antonovics (1978) have shown that callus tissue has a similar metal tolerance to that of the parent plant. They incubated callus tissue from a non-tolerant and zinc/copper-tolerant clone or Agrostis stolonifera in elevated levels of the two metals for 12 weeks. The tolerant-derived callus produced up to five times as much dry weight as that from the non-tolerant material. This response was the same regardless of whether the callus orginated from root or shoot meristems. Both tissue culture and the protoplasmic method are of importance in that tolerance is expressed at the cellular level, so allowing a measure of tolerance outside the integrity of the root-shoot system. In addition, such methods provide faster means of testing tolerance and do so using much smaller amounts of material. They also allow quantification without the total destruction of the individual.

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Cd concentration (jjg ml" ) Figure 5 The effect of increasing cadmium concentrations (\ig mL~ ) on the indices of tolerance (root elongation in +Cd solution/root elongation in -Cd solution x 100%) for a non-tolerant (o) and a cadmium-tolerant (•) population o/Holcus lanatus. The LSD at 5% level between any two means is shown. (Unpublished data of Walker, 1987.)

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Other physiological responses The inhibitory effects of heavy metal treatment on respiratory activity, photosynthetic carbon fixation and nitrogen fixation (in leguminous plants) may provide other rapid measures of differential performance between tolerant and non-tolerant genotypes. Wu, Thurman and Bradshaw (1975) demonstrated that respiration in a copper-tolerant clone of Agrostis stolonifera was less sensitive to copper than in plants from a non-tolerant clone. Thus, exposure to 10 (iM Cu for eight days resulted in only a slight drop in the respiration rate of tolerant roots, whilst there was a 25% reduction in the non-tolerant material. A copper-induced reduction in protein synthesis in the latter was also suggested. The effects of zinc treatment on fresh weight yield, photosynthetic rate and chlorophyll content served to distinguish zinc-tolerant and non-tolerant ecotypes of Silene inflata Sm. (= 5. vulgaris) in sand and solution culture (Baumeister, 1954; Baumeister and Burghardt, 1956). Whole leaf chlorophyll fluorescence and oxygen evolution in the grass Phalaris arundinacea L., measured at room temperature, have been used to assess lead and cadmium tolerance in different clones (Homer, Cotton and Evans, 1980). Measurements suggested that photosystem n was less sensitive to metal toxicities in leaf segments of tolerant clones and tolerance indices compared well with estimates of metal tolerance obtained using the conventional root elongation method. Wu and Kruckeberg (1985) found similar rates of nitrogen fixation in the nodules of copper-mine and control plants of Lotus purshianus (Benth.) Clem. & Clem, which had been shown to differ in their tolerance to copper by means of the root elongation test It appeared therefore, that tolerance was present in the legume host and its rhizobial symbiont However, N-fixing activity of another local leguminous species (Lupinus bicolor Lindl.) proved more sensitive to copper effects. There is clearly considerable scope for using other differential physiological responses in quantifying metal tolerance. Problems of Interpretation of Tolerance Measurements A number of physiological phenomena influence the interpretation of measurements of metal tolerance. Such complications give insights into the nature of plant adaptations to metal toxins and may be of major evolutionary significance. Multiple metal tolerance and co-tolerance A tacit assumption in most of the studies mentioned so far is that any specific metal tolerance has arisen as a result of the selective effects of that metal's toxicity on the plant population. It is also assumed that this is a quantitative response, in that the degree of tolerance evolved can be related directly to the intensity of the stress exerted by the metal on individuals within the population. In experimental terms, this would be demonstrated by a correlation of the mean index of tolerance with a measure of the metal's activity in the native substrate. Few workers have sought to confirm such a relationship (Wilkins, 1960; Wigham, Martin and Coughtrey, 1980; Karataglis, 1982), although it is frequently assumed. In many instances, metalliferous environments are contaminated by more than one metal in potentially toxic concentrations; heavy metals frequently co-occur in metalliferous ores. Multiple tolerance can therefore arise.

Early evolutionary studies in this field (Gregory and Bradshaw, 1965; Antonovics et al, 1971) have emphasised the independence of such tolerances and their metal specificity. However, there is now increasing evidence that in some instances plants can possess tolerance, albeit at low level, to metals which are not present at elevated concentrations in their immediate environment. This so-called co-tolerance has been shown in bacteria, fungi, algae, mosses and higher plants (Turner, 1969). Jowett (1958) studied the response of several populations of the grass Agrostis tenuis to a suite of metals. Although no actual soil analyses were presented, his published results suggest possible cases of co-tolerance. Gregory and Bradshaw (1965) repeated some of these observations regarding nickel co-tolerance in a zinc-tolerant population of the same grass. They showed the same phenomenon in populations with low soil nickel levels. There was a significant relationship between the indices of nickel and zinc tolerance but none between nickel tolerance and soil nickel concentrations. Co-tolerance was thus suggested, although Karataglis (1982) proposes that their findings may be spurious, arising from the effects of gene flow between clones of nickel-tolerant experimental plants grown alongside those tolerant to zinc and copper in the same greenhouse. There is now more convincing evidence for co-tolerance. In many of the documented instances (Allen and Sheppard, 1971; Hall, 1980; Cox and Hutchinson, 1979, 1981; Symeonidis, McNeilly and Bradshaw, 1985; Verkleij and Bast-Cramer, 1985; Verkleij et al.t 1986), co-tolerance seems to manifest itself not as full tolerance (as seen in other populations so selected), but rather as a reduced sensitivity to the metal in comparison with "control" populations. A further enigmatic subtlety of metal co-tolerance is that it is not necessarily a two-way relationship. Thus, populations of Silene cucubalus from soils enriched only with copper showed zinc co-tolerance but the converse did not apply (Verkleij and Bast-Cramer, 1985). More indirect evidence for co-tolerance includes the differential ability of plants (tolerant to metals other than those present in the native soil) to germinate and survive on other mine-spoils when compared with truly non-tolerant plants (Walley et al., 1971; Cox and Hutchinson, 1981). However, this apparent effect may again be confounded by tolerance to other stress factors associated with mine spoils. At present, there seems to be no obvious way of predicting likely metal co-tolerances from a knowledge of other tolerances, nor does there appear a rational explanation for their occurrence. There are still only relatively few reports of the phenomenon and this may either reflect reality or could, as Hall (1980) suggests, be due more to workers disbelieving or choosing to disregard any suggestions in their own data. It is rare for investigations to employ the necessary detailed experimentation to reveal any co-tolerances. Hall (1980) further suggests that a generalised decrease in sensitivity may be due to other physiological properties of the tolerant ecotype which are unrelated to metal tolerance. Several workers (Hogan and Rauser, 1979; Hall, 1980; Macnair, 1981) warn of oversimplification and failure to account for local neighbouring populations, which may be tolerant to the metals not present at the site under study, directly influencing an experimental population through the effects of gene flow. One further problem in the unequivocal demonstration of co-tolerance is the

A. J. M. Baker & P. L. Walker need to delimit metal concentrations in the substrate at which any one metal exerts a selective force. It is, however, clear that the long-held view of the specificity of metal tolerances is thrown into some question and much further work should be directed into this field of research. Constitutional tolerance The majority of experimental studies into heavy metal tolerance confirm the fundamental tenet that populations surviving in metal-contaminated habitats are differentiated from "normal" populations of the same species by the possession of genetically-based tolerances (Antonovics et al.t 1971). A number of recent studies have indicated that this is not always the case: population differentiation cannot explain the success of some species in metalliferous environments when no difference in tolerance from control material can be detected. A constitutional tolerance to metals is implied. For example, the wetland species, Typha latifolia L. (McNaughton et at., 1974; Taylor and Crowder, 1984), has been observed at sites in the vicinity of metal smelters where the substrate is heavily contaminated by metal fallout. Plants collected close to a zinc smelter were compared with controls from an unpolluted substrate by reciprocal growth experiments (McNaughton et al., 1974). There was no evidence for more tolerant genotypes in the smelter population. Other populations collected from nickeland copper-enriched soils did not exhibit the expected enhanced tolerance (Taylor and Crowder, 1984). Constitutional metal tolerance has also been suggested in the grass Andropogon virginicus Michx. (Gibson and Risser, 1982), in Thlaspi goesingense Hal csy (Reeves and Baker, 1984) and in the bryophyte, Scopelophila cataractae (Mitt.)Broth. (Shaw, 1987a). Constitutional tolerance may also exist at the generic level. Fiedler (1985) analysed five species in the genus Calochortus (Liliaceae) for nickel, cobalt and copper content. Only three were considered to be ultramafic endemics yet all showed similar metal uptake, suggesting that the ability to tolerate these metals was a constitutional property of the whole genus. Wu and Antonovics (1976) showed a differential sensitivity in the effects of roadside lead pollution on populations of Plantago lanceolata L. and Cynodon dactylon L. Comparable control and roadside populations of the latter species showed a generally higher (and similar) tolerance to lead than did the former, suggesting constitutional tolerance in C. dactylon. Antonovics (1975) attempts to explain this difference in behaviour on the basis of the greater sensitivity to lead of P. lanceolata and a reduced genotype turnover in C. dactylon during colonisation as a result of vegetative propagation. Possible evolutionary explanations for constitutional tolerance remain obscure. In the examples cited above, it is unlikely that individuals might survive (without higher tolerance) in pockets of less-toxic soil. Gibson and Risser (1982) found that elevated levels of metals were present in plants of Andropogon virginicus from a mine environment, yet' these plants displayed no greater ability to grow in metalliferous soil than plants from elsewhere. The demonstration of tolerance in other associated local species in these contrasting environments suggests that selective forces arising from metal toxicity are strong enough to bring about population differentiation in "sensitive" species (Wu and Antonovics, 1976). Species thus clearly differ in these critical thresholds, an

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alternative interpretation of constitutional tolerance. Reeves and Baker (1984) proposed that a non-specific detoxification system in T. goesingense might explain the similar characteristics of zinc, nickel and cobalt uptake in serpentine and calcareous populations of this species. Fiedler (1985) suggested that the Calochortus spp. she studied may be fortuitously tolerant as a coincidence of adaptation to other stress factors. Whatever the reasons, it is clear that the original concept of metal-specific tolerances evolving at the level of an ecotype cannot always be invoked to explain the success of all species in contaminated environments. Stimulatory responses For most heavy metals, a dose-response curve similar to the generalised scheme presented in Figure 1 can be constructed in order to assess impact on plant growth. The shape of this curve will depend on both metal essentiality/toxicity and species sensitivity. A feature of many such response curves, even for non-essential heavy metals, is a stimulatory response in growth or yield at very low external concentrations, followed by inhibitory effects at higher concentrations. Many workers have not detected this low-level stimulatory response because of the metal concentrations chosen in tolerance tests. However, there is some evidence to suggest that in tolerant plants the metal concentrations producing both stimulatory and inhibitory effects are higher than for normal (non-tolerant) plants. The effect is manifested by an apparent "need" for otherwise toxic metals, but in reality the response curve has been shifted up-scale towards higher treatment concentrations. Antonovics et al. (1971) detail several reports of stimulatory effects and in many instances the enhancement of growth is small and not significantly greater than values in control treatments, but its widespread occurrence merits further investigation. Some reports, however, suggest large effects; the most extreme of these being a 1,000% increase in biomass (above control) in zinc-tolerant Thlaspi alpestre L. grown in a zinc-amended culture solution (Mathys, 1980). Whilst most of the reports of stimulatory responses are for essential micronutrients, similar responses have been detected for metals which are not known to have any essential physiological role. The response of the grass Holcus lanatus to cadmium has been investigated by several workers (Coughtrey and Martin, 1977; Brown, 1983; Walker, 1987) and stimulation of root growth has been demonstrated in cadmium-tolerant individuals exposed to low-levels of the metal (Figure 5). Soil culture experiments have also demonstrated enhanced growth of tolerant plants. Shaw (1987a) found that eight populations of the moss Scopelophila cataractae grew significantly better on a mine spoil than on a control soil medium. The effect was unrelated to the metal contents of the sites of origin of the moss populations. Similarly, a copper/nickel-tolerant population of the grass Deschampsia cespitosa had a higher germination and survival rate on its native, contaminated soil than on a control medium (Cox, 1979; Cox and Hutchinson, 1981). Possible explanations for these stimulatory responses in tolerant plants may well be found when the metal tolerance mechanisms involved are understood more fully. It is unlikely, however, that tolerant plants have greater absolute requirements of essential trace metals. It is more probable that the efficiency of the tolerance mechanism in detoxifying the metal may be so great that it prevents the metal from being internally "available"

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unless external concentrations are sufficiently high. Such a hypothesis has been proposed to explain the observed stimulatory effects on root growth of both iron and aluminium in calcifuge plants (Grime and Hodgson, 1969). Similar effects for both the essential (iron) and non-essential (aluminium) metals were thought indicative of a non-specific detoxification system involved in tolerance to polyvalent cations. It can be argued that physiological requirements for iron are therefore elevated in calcifuge plants because of the efficiency of this detoxification system. A similar theory could explain stimulatory responses to zinc and copper in zinc- and copper-tolerant plants, but cannot be invoked for lead and cadmium responses. Explanations of the stimulatory effects of non-essential metals are yet to be found and further research in this area should be rewarding. The responses referred to above are important in that they may confound "conventional" methods of tolerance assessment when growth in a treatment solution is expressed as a fraction of sub-optimal growth in a control (Wilkins, 1978). To overcome this problem the inclusion of an additional control containing a slightly elevated level of the toxin has been suggested (Wilkins, 1978), but it is difficult to define an appropriate background metal concentration. Indudble metal tolerance Since the demonstration of metal tolerance in higher plants by Bradshaw in 1952, most workers have assumed, if not demonstrated unequivocally, that tolerance is an immutable,

specific character with a high degree of heritability, free from any apparent Lamarckian influences. In genecological experiments on metal tolerance, individuals are sampled from populations and are invariably "grown-on" in a potting compost under standardised environmental conditions to avoid so-called "carry-over" effects. This practice is often used as a means of ensuring an identical starting point for plants under study. Furthermore, observations by several experimenters (Bradshaw, 1952; Gregory and Bradshaw, 1965) suggest that growth in a normal, uncontaminated soil does not reduce the phenotypic expression of heavy metal tolerance. Wilkins (1978) comments explicitly on the absence of evidence showing that tolerance can be altered by prior treatments. However, Repp (1973) believed that stress resistance comprised both genotypic and phenotypic components; the former can only be altered through selection, whilst the latter represent a range of physiological adaptations within boundaries defined by the genotype. This implies that changes during one life history can and do occur in response to environmental influences. In the light of new evidence a reappraisal of the plasticity of metal tolerance would now seem appropriate. Coughtrey (1978) first investigated the effects of pretreating seedlings of Holcus lanatus by exposing them to a low-level of metal prior to a more toxic dose. Brown and Martin (1981) later performed a more detailed examination of pretreatment effects on a cadmium-tolerant and a non-tolerant population of this grass. They found that an increase in cadmium tolerance could be induced in both populations by a

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Populations Figure 6 Parallel indices of cadmium tolerance for populations of the grasses Holcus lanatus, Deschampsia cespitosa, Festuca rubra and Agrostis capillaris, grown in their own soil (open bars), or potting compost (shaded bars), for three years and measured at 1 jig mL" cadmium. Key to populations: H, Hallen Wood (aerially-polluted site); S, Shipham (calamine workings); V, Velvet Bottom (lead smelter); W, Wetmoor (unpolluted control). (Redrawn from Baker et al., 1986, by kind permission of the New Phytologist Trust.)

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A. /. M. Baker & P. L. Walker previous low-level exposure to the metal. The effect on root elongation was exclusive to those roots which had been pretreated. Shaw (1987b) has performed similar experiments using the moss Funaria hygrometrica Hedw. In examining pretreatments with zinc and copper, he found that differences in tolerance were in the main genetically-based. Some pretreatment effects did occur although responses were irregular and non-specific. Results suggesting similar changes in metal tolerance have been seen in H. lanatus by the authors. Short-term metal pretreatment effects have also been demonstrated in the lichen genus Peltigera (Beckett and Brown, 1983) where it was found that pretreating discs of P. membranacea (Ach.)Nyl. with zinc, decreased the inhibition of photosynthesis by both zinc and cadmium. Watkins (1985) was unable to detect similar responses in Agrostis capillaris. In two experiments in which plants were preconditioned by growth in normal or copper-mine soils, only one anomalous effect was detected; the mine-spoil treated plants proved to be less arsenic tolerant than their normal soil counterparts (A. Watkins, personal communication, 1986). Longer term effects are also known. Lepp and Dickinson (1986) found differential sensitivity to copper in two stands of the same cultivar of Coffea arabica. The greater tolerance of one stand may have been due to it receiving copper-based fungicide sprays over a number of years. Tolerance can also be "lost". Stokes (1975) reported the loss of copper tolerance in an isolate of Scenedesmus acutiformis var. alternant. She found that excluding copper from the culture medium for more than one week resulted in a significant reduction in copper tolerance but did not alter the response to nickel. The loss could be "corrected" to almost original levels after re-exposing the isolate to copper for only 12 days. Baker et al. (1986) have found that the soil used for raising populations of H. lanatus can influence the degree of cadmium tolerance measured after a period of cultivation. The main effects included some loss of cadmium tolerance in a smelter population when grown on uncontaminated soil. Conversely, a non-tolerant population showed some induction of tolerance when grown on soils containing cadmium. The tolerance of a mine-spoil population was, however, largely unaffected, suggesting that different populations may differ in the extent of any plastic responses. Further experiments with Agrostis capillaris, Deschampsia cespitosa and Festuca rubra also revealed a general plasticity in cadmium tolerance (Figure 6). There would now appear to be some evidence, albeit tenuous, linking the plasticity in tolerance to the nature of contamination. Baker et al. (1986) found that their smelter population of//, lanatus showed more plasticity in its cadmium tolerance than did a highly tolerant mine-spoil population. It was suggested that this may be a result of the smelter population originally being non-tolerant. The development of tolerance would have been slow, perhaps starting as selection for plasticity to cadmium sensitivity. Only when the soil cadmium concentrations resulting from deposition from the smelter had reached phytotoxic levels (equivalent to those of mine sites) would selective forces be strong enough to effect genotypic changes in the population. By comparison, mine sites represent a more stable and continuously toxic environment where selective forces have been strong for many years and the effects of selection are clear-cut (Baker, 1987). In support of this theory evidence of the rapid appearance of small, yet significant

increases in cadmium tolerance of H. lanatus in artificially-contaminated plots has already been demonstrated (Coughtrey, Martin and Shales, 1978). From the experiments discussed above, it is apparent that the growth media used to propagate plants prior to tolerance testing may influence the extent of plant response. This will be especially important in studies comparing the responses of various tolerant populations where ecotypic differences may be small. Despite these caveats, few workers use native soils to prepare plants for such experiments. The extent to which tolerance may be plastic therefore remains largely untested and research in this area is urgently needed. References Allen, W.R. and Sheppard, P.M. 1971. Copper tolerance in some Californian populations of the monkey flower Mimulus guttatus. Proc. R. Soc. Lond., Ser. B, 177, 177-196. Antonovics, J. 1975. Metal tolerance in plants: perfecting an evolutionary paradigm. In: Proc. 1st. Int. Conf. Heavy Metals in the Environment, Vol.2, pp.169-186. Toronto, University of Toronto, Canada. Antonovics, J., Bradshaw, A.D. and Turner, R.G. 1971. Heavy metal tolerance in plants. Adv. Ecol. Res., 7,1-85. Baker, A.J.M. 1987. Metal tolerance. New Phytol., 106 (Suppl.), 93-111. Baker, AJLM. and Walker, P.L. 1989. Ecophysiology of metal uptake by tolerant plants. In: Shaw, J. (ed.), Heavy Metal Tolerance in Plants. CRC Press Inc., New York (in press). Baker, A.J.M., Grant, C.J., Martin, M.H., Shaw, S.C. and Whitebrook, J. 1986. Induction and loss of cadmium tolerance in Holcus lanatus L. and other grasses. New Phytol., 102, 575-587. Baumeister, W. 1954. Uber den Einfluss des Zinks bei Silene inflata Smith. I. Berichte der Deutschen botanischen Gesellschaft, 67, 205-213. Baumeister, W. and Burghardt, H. 1956, Uber den Einfluss des Zinks bei Silene inflata Smith. EL CQz Assimilation und PigmentgehalL Berichte der Deutschen botanischen Gesellschaft, 69,161-168. Beckett, R.P. and Brown, D.H. 1983. Natural and experimentally-induced zinc and copper resistance in the lichen genus Peltigera. Annals Bot., 52, 43-50. Berry, W.L. and Wallace, A. 1981. Toxicity: the concept and relationship to the dose response curve. /. Plant Nutrit., 3, 13-19. Bradshaw, A.D. 1952. Populations of Agrostis tenuis resistant to lead and zinc poisoning. Nature, 169, 1098. Bradshaw, A.D. 1984. Adaptation of plants to soils containing toxic metals - a test for conceit. In: Evered, D. and Collins, G.M. (eds.), Origins and Development of Adaptation, pp.4-19. CIBA Foundation Symposium No.102, Pitman, London. Briggs, D. 1972. Population differentiation in Marchantia polymorpha L. in various lead pollution levels. Nature, 238, 166-167. Brown, H. 1983. A Study of Cadmium Tolerance in the Grass Holcus lanatus L. PhD Thesis, University of Bristol. Brown, H. and Martin, M.H. 1981. Pretreatment effects of cadmium on the root growth of Holcus lanatus L. New Phytol., 89, 621-629. Clarkson, D.T. 1965. The effect of aluminium and other bivalent metal cations on cell division in the root apices ofAllium cepa. Annals Bot., 29,309-315. Cook, S., Lefebvre, C. and McNeilly, T. 1972. Competition between metal tolerant and normal plant populations on normal soil. Evolution, 26, 366-372.

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Coughtrey, PJ. 1978. Cadmium in Terrestrial Ecosystems - a Case Study at Avonmouth, Bristol. PhD Thesis, University of Bristol. Coughtrey, P.J. and Martin, M.H. 1977. Cadmium tolerance of Holcus lanatus from a site contaminated by aerial fallout. New Phytol., 79, 273-280. Coughtrey, P.J., Martin, M.H. and Shales, S.W. 1978. Preliminary observations on cadmium tolerance in Holcus lanatus. L. from soils artificially contaminated with heavy metals. Chemosphere, 2, 193-198. Cox, R.M. 1979. Multiple tolerance relations in native plants and their applications to reclamation. In: Perry, R. (ed.), Proc. International Conference, Management and Control of Heavy Metals in the Environment, London, pp.202-205. CEP Consultants Ltd., Edinburgh. Cox, R.M. and Hutchinson, T.C., 1979. Metal co-tolerances in the grass Deschampsia cespitosa. Nature, 279, 231-233. Cox, R. and Hutchinson, T.C. 1981. Multiple and co-tolerance in the grass Deschampsia cespitosa: adaptation, pre-adaptation and "cost". J. Plant Nutrit., 3, 731-741. Ernst, W. 1972. Ecophysiological studies on heavy metal plants in South Central Africa. Kir Ida, 8, 125-145. Ernst, W. 1976. Physiological and biochemical aspects of metal tolerance. In: Mansfield, T.A. (ed.), Effects of Air Pollutants on Plants, pp.115-133. Cambridge University Press, Cambridge. Fiedler, P.L. 1985. Heavy metal accumulation and the nature of edaphic endemism in the genus Calochortus (Liliaceae). Am. J. Bot., 72, 1712-1718. Gerakis, P.A., Veresoglou, D.S. and Sakellariadis, S.D. 1980. Differential response of sugar beet Beta vulgar is L. cultivars to lead. Envir. Pollut. Ser. A., 21, 77-83. Gibson, DJ. and Risser, P.G. 1982. Evidence for the absence of ecotypic development in Andropogon virginicus L. on metalliferous mine wastes. New Phytol., 92, 589-599. Gregory, R.P.G. and Bradshaw, A.D. 1965. Heavy metal tolerance in populations of Agrostis tenuis Sibth. and other grasses. New Phytol, 64, 131-143. Cries, B. 1966. Zellphysiologische Untersuchungen uber die Zinkresistenz bei Galmeiokotypen und Normalformen von Silene cucubalus Wib. Flora, 156, 271-290. Grime, J.P. and Hodgson, J.G. 1969. An investigation of the ecological significance of lime-chlorosis by means of large-scale comparative experiments, pp.67-99. In: Rorison, I.H. (ed.), Ecological Aspects of the Mineral Nutrition of Plants. British Ecological Society Symposium No.9, Blackwell Scientific Publications, Oxford. Hall, A. 1980. Heavy metal co-tolerance in a copper-tolerant population of the marine fouling alga, Ectocarpus siliculosus (Dillw.) Lyngbye. New Phytol., 85, 73-78. Hertstein, U. and Jager, H-J. 1986. Tolerances of different populations of three grass species to cadmium and other metals. Envir. Expl. Bot., 26, 309-319. Hogan, G.D. and Rauser, W.E. 1979. Tolerance and toxicity of cobalt, copper, nickel and zinc in clones of Agrostis gigantea. New Phytol., 83, 665-670. Homer, J.R., Cotton, R. and Evans, H.E. 1980. Whole leaf fluorescence as a technique for measurement of tolerance of plants to heavy metals. Oecologia (Berl.), 45, 88-89. Humphreys, M.O. and Nicholls, M.K. 1984. Relationships between tolerance to heavy metals in Agrostis capillaris L. (A. tenuis Sibth.). New Phytol., 98, 177-190. Ingrouille, M.J. and Smirnoff, N. 1986. Thlaspi caerulescens J. and C. Presl. (T. alpestre L.) in Britain. New Phytol, 102, 219-233. Johnston, W.R. and Proctor, J. 1981. Growth of serpentine and non-serpentine races of Festuca rubra in solutions simulating the chemical conditions in a toxic serpentine soil. /. Ecol., 69, 855-869.

Jowett, D. 1958. Populations of Agrostis spp. tolerant to heavy metals. Nature, 182, 816-817. Jowett, D. 1964. Population studies on lead tolerant Agrostis tenuis. Evolution, 18, 70-81. Karataglis, S.S. 1980. Selective adaptation to copper of populations of Agrostis tenuis and Festuca rubra (Poaceae). Plant Systematics and Evolution, 134, 215-228. Karataglis, S.S. 1982. Combined tolerance to copper, zinc and lead by populations of Agrostis tenuis. Oikos, 38, 234-241. Lepp, N.W. and Dickinson, N.W. 1986. Effect of copper on germination of Coffea pollen: Possible induction for copper tolerance. In: Proc. 2nd. Int. Conf. Environmental Contamination, Amsterdam, pp.33-35. CEP Consultants, Edinburgh. Macnair, M.R. 1981. Tolerance of higher plants to toxic materials. In: Bishop, J.A. and Cooke, L.M. (eds.), Genetic Consequences of Man Made Change, pp.177-207. Academic Press, New York and London. Maschmeyer, J.R. and Quinn, J.A. 1976. Copper tolerance in New Jersey populations of Agrostis stolonifera and Paronychia fastigiata. Bulletin of the Torrey Botanical Club, 103, 244-251. Mathys, W. 1977. The role of malate, oxalate and mustard oil glucosides in the evolution of zinc-resistance in herbage plants. Physiol. Plant., 40, 130-136. Mathys, W. 1980. Zinc tolerance by plants. In: Nriagu, J.O. (ed.), Zinc in the Environment. Part II: Health Effects, Chap.17. John Wiley & Sons Inc., New York. McNaughton, S.J., Folsom, T.C., Lee, T., Park, F., Price, C., Roeder, D., Schmitz, J. and Stockwell, C. 1974. Heavy metal tolerance in Typha latifolia without the evolution of tolerant races. Ecology, 55, 1163-1165. Powell, M.J., Davies, M.S. and Francis, D. 1986. The influence of zinc on the cell cycle in the root meristem of a zinc-tolerant and a non-tolerant cultivar of Festuca rubra L. New Phytol., 102, 419-428. Reeves, R.D. and Baker, A.J.M. 1984. Studies on metal uptake by plants from serpentine and non-serpentine populations of Thlaspi goesingense Halacsy (Cruciferae). New Phytol., 98, 191-204. Repp, G. 1963. Die Kupferresistenz des Protoplasmas hoherer Pflanzen auf Kupferzbo'den. Protoplasma, 57, 643-659. Repp, G. 1973. Cytoecological investigations with regard to the mechanism of chemical resistance of plants. In: Hudiik, R.J. and Davis, G. (eds.), Ecology of Devastated Land, Vol.1, pp.445-466. Gordon & Breach, New York and London. Russell, G. and Morris, O.P. 1970. Copper tolerance in the marine fouling alga Ectocarpus siliculosus. Nature, 228, 288-289. Schiller, W. 1974. Verusche zur Kupferresistenz bei Schwermetallokotypen von Silene cucubalus Wib. Flora, 163, 327-341. Searcy, K.B. and Mulcahy, D.L. 1985a. Pollen selection and the gametophytic expression of metal tolerance in Silene dioica (Caryophyllaceae) and Mimulus guttatus (Scrophulariaceae). Am. J. Bot., 72, 1700-1706. Searcy, K.B. and Mulcahy, D.L. 1985b. The parallel expression of metal tolerance in pollen and sporophytes of Silene dioica (L.) Clairv., 5. alba (Mill.) Krause and Mimulus guttatus DC. Theor. Appl. Genetics, 69, 597-602. Shaw, J. 1987a. Evaluation of heavy metal tolerance in Bryophytes n. An ecological and experimental investigation of the "copper moss", Scopelophila cataractae (Pottiaceae). Am. J. Bot., 74, 813-821. Shaw, J. 1987b. Effect of environmental pretreatment on tolerance to copper and zinc in the moss Funaria hygrometrica. Am. J. Bot., 74, 1466-1475. Stokes, P.M. 1975. Adaptation of green algae to high levels of

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