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Heavy metals derived from land-based mining, industrial and agricultural activities and transported via the atmosphere and rivers can ultimately reach coastal and estuarine waters, where they accumulate within the biota, including marine macrophytes (DeGregory et al., 1996; Marsden & DeWreede, 2000; Grout & Levrings, 2001; Stauber et al., 2001). In oceanic surface waters the total copper concentration ([CuT]) is maintained at pm to nm levels and is largely governed by biological activity (Bruland, 1980; Coale & Bruland, 1990; Sunda & Huntsman, 1995). By comparison, the [CuT] in Restronguet Creek (Fig. 1), part of the Fal Estuary system (south-west UK) into which mine drainage water flows, is 0.2 m (daily average), a value 20-fold higher than that of uncontaminated estuaries (Bryan & Langston, 1992; The Environment Agency, UK, unpubl. data).
Figure 1. Map of the sampling area in south-west England, UK, showing the Ordnance Survey (OS) grid references of the sampling sites.
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From the considerable body of literature amassed on metal tolerance in higher plants, it is evident that plants growing on metalliferous soils can evolve resistance to the normally toxic effects of metals and that certain species have the capacity to develop ecotypes expressing different levels of tolerance (Ernst et al., 1992; van Hoof et al., 2001). For marine macroalgae (seaweeds), similar data are much less extensive (Brown & Depledge, 1990), with much of the research restricted to the evaluation of hyperaccumulation of metal ions for biomonitoring purposes (Phillips, 1990; Riget et al., 1995; Gibb et al., 1996).
Copper is considered one of the most toxic heavy metal ions to algae and plants and is a potent inhibitor of photosynthesis. The results from several studies on microalgae and plants suggest that excess Cu2+ has a complex toxic action on the primary reactions of photosynthesis and that the inhibitory effect on photosynthetic electron transport is targeted at photosystem II (PSII) (Schröder et al., 1994; Cid et al., 1995; Jegerschöld et al., 1995; Yruela et al., 1996). However, detailed investigations of Cu2+-induced damage to the photosynthetic apparatus of brown macroalgae are limited (Küpper et al., 2002).
The ability of brown algae to resist elevated external Cu2+ concentration ([Cu2+]ext) is, at least in part, related to the high levels of polyphenolics that sequester metal ions in specialized vesicles (physodes) within cells close to the thallus surface (Lignell et al., 1982; Smith et al., 1986; Stengel & Dring, 2000). It may also be dependent on the exudation of metal complexing ligands that alter the speciation of copper in the surrounding water or act as a detoxification mechanism (Sueur et al., 1982; Gledhill et al., 1999). Recent studies on the genus Fucus have provided detailed information on the toxicity of Cu2+ to growth and development of zygotes (Andersson & Kautsky, 1996; Bond et al., 1999; Gledhill et al., 1999), and the mechanisms of toxicity in Fucus development are becoming better understood (Nielsen et al., 2003). However, few studies have directly compared the physiological responses of adult individuals from populations that are exposed to elevated concentrations of metals in their natural environment with those that are not (Bryan & Gibbs, 1983). As a consequence, information on differential levels of tolerance of populations and the mechanisms by which this may be achieved is lacking.
In this study, we have exploited the natural resistance of Fucus populations to copper contamination in order to derive further insight into toxicity and tolerance mechanisms. We assess the degree of Cu2+ tolerance of three populations of Fucus serratus collected from sites with different levels of copper contamination by measuring the growth of adults and embryos, and the accumulation of Cu2+ by adults. Furthermore, information on the mode of action of Cu2+-induced damage on the photosynthetic apparatus of adults is provided by measuring chlorophyll fluorescence parameters as well as oxygen evolution and consumption.
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We have identified interpopulation differences in the response of F. serratus to Cu2+ that are related to the copper status of their natural environment. The physiological responses of F. serratus to Cu2+ exposure identify the Restronguet population as highly resistant, whereas the populations from Bantham and Wembury, which were very sensitive to Cu2+ exposure, are classed as nonresistant. These results support those from a study by Bryan & Gibbs (1983) who showed that RGRs of adult F. serratus from Restronguet Creek were unaffected by concentrations up to 1 m CuT, a value two- to five-fold higher than that tolerated by F. serratus from uncontaminated sites. Moreover, our results imply that Cu2+ resistance in F. serratus is an inherited character. Progeny derived from individuals growing in Restronguet Creek displayed significantly higher levels of resistance than those obtained from the other two sites, and the degree of resistance in the offspring was similar to those of adults.
Few studies have addressed the mechanisms of resistance of seaweeds to metals, although both avoidance and tolerance have been observed (Hall et al., 1979; Correa et al., 1996). Exposure to Cu2+ has been shown to induce the expression of a metallothionein-encoding gene in Fucus (Morris et al., 1999), although metal tolerance of brown algae is also closely related to internal and external complexation with ligands such as polyphenols (Smith et al., 1986; Gledhill et al., 1999). The ability of brown algae to hyperaccumulate metal ions in a concentration-dependent manner (Stengel & Dring, 2000) is often used in biomonitoring programmes. Here, we show that copper accumulation in Fucus is not only dependent on the [Cu2+]ext to which they are exposed but also on the contamination history of their respective locations, with the Restronguet material accumulating only half the concentration of Bantham and Wembury during exposure to high but ecologically relevant [Cu2+]ext. Exclusive use of local material in biomonitoring programmes may therefore underestimate the actual pollution pressure in contaminated habitats. The results also suggest that Cu2+-tolerant populations may, at least in part, rely on an exclusion mechanism. The lower accumulation ability of Wembury material at [Cu2+]ext up to 211 nm is likely to be related to the reduced surface area : volume ratio of these algae, which may be a response to greater wave exposure at this location (pers. obs.). Irrespective of the capacity of brown algae to sequester heavy metals (Lignell et al., 1982; Smith et al., 1986; Gledhill et al., 1999), cytosolic or organellar [Cu2+] may increase beyond cellular control and reach levels that interfere with metabolic processes. It is well-known that Cu2+ is a potent inhibitor of photosynthesis in both higher plants (Yruela et al., 1996; Ouzounidou et al., 1997) and brown algae (Plötz, 1991; Küpper et al., 2002).
The data presented here also suggest that the greater susceptibility of nontolerant algae to Cu2+ toxicity is in part due to the photosynthetic apparatus being less resistant than that of tolerant algae. Exposure to Cu2+ may result in extensive and irreversible damage to light-harvesting chlorophyll, indicated by the large decrease in Fo and Fm in nontolerant, but not tolerant, fronds of Fucus. Similar results have been observed in the brown alga Ectocarpus siliculosus (Küpper et al., 2002) and wheat seedlings (Ciscato et al., 1997). Decreased Fo and Fm in response to Cu2+ exposure is reportedly associated with chlorophyll degradation (Ciscato et al., 1997) or substitution of magnesium (Mg2+) by Cu2+ in the chlorophyll molecule (Küpper et al., 2002). However, for Restronguet fronds, maintenance of Fm and Fo at high [Cu2+] suggests that neither chlorophyll degradation nor Cu2+ substitution of Mg2+ occurs in tolerant fronds during Cu2+ exposure. The observed Cu2+-dependent increase in Fv/Fm in nontolerant fronds may be related to increased photosynthetic efficiency of the remaining undamaged photosynthetic units satisfying a continued demand from downstream photosynthetic processes, which may have been affected to a lesser degree than light harvesting. In contrast, dark-adapted Fv/Fm of tolerant fronds was largely unaffected by Cu2+. Similar responses have also been observed in other brown and green algae (Küpper et al., 2002) and seedlings of maize and wheat (Ciscato et al., 1997; Ouzounidou et al., 1997).
The effects of Cu2+ on photosynthetic fluorescence parameters suggest that Cu2+ tolerance in Fucus may be related to processes similar to those involved in light adaptation. Under photosynthetically saturating light conditions, exposure to Cu2+ resulted in a reduction in the quantum yield of photosystem II (ΦPSII) of tolerant fronds. A similar response has been shown in higher plant tissue under Cu2+ exposure (Yruela et al., 1996; Ciscato et al., 1997; Pätsikkäet al., 1998), and is normally associated with inhibitory effects of Cu2+ on plastoquinone a (QA) reduction and/or interference with electron donation to photochemical reactions in PSII downstream of QA (Yruela et al., 1991, 1993, 1996; Schröder et al., 1994; Jegerschöld et al., 1995). Alternatively, Cu2+ may target chloroplast membrane H+ATPases, thus lowering the demand for H+ and electron transport, indirectly resulting in excitation energy entrapment in PSII. Inhibitory effects of Cu2+ on ATPases and ion channels are known from several plant and animal systems (Osipenko et al., 1992; Demidchik et al., 1997, 2001). In tolerant Fucus fronds, Cu2+-induced reduction of light-saturated ΦPSII coincided with increased NPQ, which is normally associated with xanthophyll cycle-dependent energy dissipation in brown algae (Harker et al., 1999; Vershinin & Kamnev, 1996; Coelho et al., 2001) and higher plants (Demmig-Adams, 1998; Ruban & Horton, 1999; Ebbert et al., 2001). Excitation energy trapped within PSII in tolerant fronds exposed to Cu2+ appears to be dissipated through quenching mechanisms analogous to those relieving light-induced photoinhibition. Failure of nontolerant fronds to disperse or relieve excess supply of electrons in response to inhibition of electron transport may reflect a smaller or less robust pool of xanthophylls. Moreover, excess excitation energy may induce production of reactive oxygen species (ROS) (Sandmann & Böger, 1980; Jegerschöld et al., 1995; Yruela et al., 1996), leading to disruption of photosynthetic units and chlorophyll a breakdown.
Physiological responses of F. serratus exposed to Cu2+, as well as the mechanism by which the metal is tolerated, are complex. Although metal exclusion mechanisms clearly are involved in Cu2+ tolerance in Fucus, the Cu2+ resistance of the photosynthetic apparatus may also be a significant factor. Therefore, studies of toxicity effects on isolated chloroplasts from the different populations would provide further information on Cu2+ tolerance at the cellular level. Furthermore, Cu2+ tolerance mechanisms in this species may involve a genetic component, giving rise to tolerance early in zygote development (Nielsen et al., 2003). However, Cu2+ tolerance may also be affected by factors other than the contamination history of the habitat. For example, the influence of the light climate and macro nutrient availability on physiological and biochemical responses in brown algae exposed to Cu2+ is not fully understood and future work should address interactive effects between these parameters.