Author for correspondence: Hanne D. Nielsen Tel: +45 46 71 27 85 Fax: +45 46 71 30 11 Email: hnielsen@Ruc.dk
• A comparative study of copper (Cu) toxicity and tolerance in three populations of Fucus serratus was conducted by examining Cu2+ effects on various physiological parameters.
• Chlorophyll fluorescence, oxygen evolution, copper content, and relative growth rate of embryos and adults were measured on Cu2+-exposed material.
• Algae naturally exposed to elevated total Cu concentration (CuT), were more Cu2+ resistant than those from clean sites, as indicated by higher embryo and adult growth rates and lower copper contents. The Cu2+ tolerance of F. serratus is at least partly inherited and relies partly on metal exclusion.
• There were inhibitory effects of Cu2+ on oxygen exchange rates in both tolerant and non-tolerant algae. By contrast to sensitive algae, the maximum efficiency of photosystem II (Fv/Fm), maximum fluorescence (Fm) and zero fluorescence (Fo) of resistant algae were unaffected by Cu2+, whereas decreased quantum yield (ΦPSII) and increased nonphotochemical quenching (NPQ) were most pronounced in resistant algae. Inhibitory effects of Cu2+ on ΦPSII may result in the excitation energy being dissipated through xanthophyll-dependent quenching mechanisms in tolerant algae. In nontolerant algae, lower energy dissipation may result in chlorophyll degradation.
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.
Materials and Methods
Algal material and general culture conditions
Individual F. serratus (L.) plants bearing vegetative fronds and mature receptacles were collected from three populations in south-west England, UK (Fig. 1). Algae collected from Restronguet Creek (Fal Estuary) are subjected to anthropogenic copper pollution in their natural environment (as high as 0.5–1 m CuT), whereas those collected from Bantham Quay (Avon Estuary) and Wembury Beach are exposed to natural background estuarine and coastal concentrations of copper, of about 10 nm CuT (Bryan & Langston, 1992; The Environment Agency, UK, unpubl. data).
Algae were rinsed in filtered (0.45 m cellulose nitrate membrane) natural seawater (FSW). To ensure experimental material of similar age, vegetative tips (approx. 3 cm) were cut from fronds and allowed to recover in FSW from their respective locations for 8 d, during which time FSW was changed daily. Frond tips were maintained at 15°C under 250 mol m−2 s−1 photosynthetic active radiation (PAR) provided by fluorescent lamps (Phillips ‘cool white’) on a 16 h/8 h light/dark cycle. Frond tips were transferred to individual beakers containing 100 ml artificial seawater culture medium (ASW) based on Aquil (Morel et al., 1979) but without the addition of a chelating agent (nitrilotriacetic acid (NTA) or ethylenediaminetetraacetic acid (EDTA)) as this has been found to result in a small reduction in growth when compared with unchelated total copper concentrations (Gledhill et al., 1999). For experimental purposes, ASW was enriched with CuSO4·H2O to yield [CuT] ranging between 0 and 2 m. Initial, nominal, concentrations of Cu2+ in ASW were calculated by applying the total concentrations of the ASW constituents to the chemical equilibrium-modelling program mineql version 2.1 (Westall et al., 1976). Fucus is known to release metal-complexing ligands when exposed to elevated concentrations of metal ions, which bring about a decrease in the [Cu2+]ext in the culture medium (Gledhill et al., 1999). To compensate for ligand release, ASW was changed daily during experimentation. Measurements of relative growth rate (RGR), photosynthetic parameters and copper accumulation were determined after 12 or 23 d exposure to [Cu2+]ext of 0 (control), 42.2, 211, 422 and 844 nm, under the temperature and light regime described earlier. To provide information on the inheritance of Cu2+ resistance, the effect of Cu2+ on embryo rhizoid elongation was also studied.
Effects of Cu2+ on algal growth
Growth measurements of adults Five replicates of each population were exposed to each of the five treatments (k = 5, n = 5). Daily RGRs of vegetative apical tips were calculated using the formula:
(Wf and Wi are the final and initial f. wt (measured to an accuracy of 0.1 mg), respectively; d is the length of exposure in days).
Growth measurements of embryos Upon germination, Fucus embryos produce a single rhizoid, the elongation of which increases the total body length several-fold within a few days (Nielsen et al., 2003). Embryo growth was determined by measuring rhizoid elongation of germinated zygotes acquired from adult material from each of the three populations. Gametes were released from mature receptacles and fertilization was achieved according to the methods of Nielsen et al. (2003). Embryos were sown at a density of approx. 100 per cm2 onto small Petri dishes (2.5 cm diameter) with coverslip bases and incubated at 15°C under 60 mol m−2 s−1 unidirectional white light (‘cool white’ fluorescent lamps). Embryos were allowed to develop in Cu2+-free ASW until 18 h after fertilization and subsequently transferred to ASW containing 84.4, 211 or 844 nm Cu2+ or retained in Cu2+-free medium and grown for a total of 10 d. Rhizoid length, defined as the distance from the rhizoid/thallus dividing cell wall to the rhizoid tip, was recorded digitally from bright field images and was measured using analytical software (lucida; Kinetic Imaging, Liverpool, UK). Three replicate dishes of each population were exposed to each of the four treatments (k = 4, n = 3). Growth is expressed as the average RGR of rhizoid elongation of 25 embryos in each dish.
Copper content of fronds
After 23 d of exposure, the total copper content of all apical tips was measured by atomic absorption spectrophotometry (AAS). Material was rinsed thoroughly in nano-pure water, frozen at −20°C and freeze-dried (Super Modulyo freeze-drier; Girovac, North Walsham, UK) for 24 h. Freeze-dried and homogenized material (approx. 0.1 g) was microwave digested (CEM-2000; CEM Microwave Technology, Birmingham, UK) in 2 ml conc. HNO3 for 35 min and subsequently made up to 5 ml volume with nano-pure water and analysed for copper in a Varian 600 series AAS (Varian Ltd, Walton-on-Thames, UK) in graphite furnace mode. Copper standards were made using a certified copper standard solution (Merk, Lutterworth, UK) acidified to the same pH as the samples with HNO3.
Effects of Cu2+ on the photosynthetic apparatus
Chlorophyll fluorescence measurements In vitro chlorophyll fluorescence of apical tips exposed to the different copper treatments was measured after 12 d and 23 d with a modulated fluorescence monitoring system (Hansatech Instruments, Norfolk, UK) based on the principles described by Schreiber et al. (1986) and carried out by application of the rapid light curve technique (RLC) (White & Critchley, 1999) to the apical part of the front tips. Individual tips were removed from the incubation medium and placed in a holder at a fixed distance from the measuring fibre at an ambient temperature of 15°C. Dark adaptation was ensured by application of a 5.5-s far-red light pulse (6 mol m−2 s−1), which drained electrons from PSII and ensured its full relaxation. Zero fluorescence (Fo) was measured and followed by application of a 0.8-s light pulse (3000 mol m−2 s−1) and measurement of maximum fluorescence (Fm). Subsequently, actinic light was switched on and the light level gradually increased. Each step lasted 30 s, followed by a 0.8-s saturating light pulse and measurement of the maximum fluorescence in the light (Fm′) and steady-state fluorescence in the light (Ft). From light response curves it was established that a photosynthetic photon fluence rate (PPFR) of 365 mol m−2 s−1 saturated Fucus photosynthesis. Standard total recording time of a standard RLC was 7 min. The maximum efficiency of PSII in the dark-adapted state, the maximum efficiency of PSII, was calculated as:
(δF is the difference between the maximum fluorescence, Fm′, of light adapted algae and the steady-state fluorescence, Ft). Changes in chlorophyll fluorescence due to changes in nonphotochemical quenching (NPQ) were calculated according to (Fm − Fm′)/Fm′ (Maxwell & Johnson, 2000). The experimental design for chlorophyll fluorescence followed that of the adult growth procedure of k = 5, n = 5.
Oxygen consumption and evolution
After 23 d exposure to the various copper treatments gross photosynthesis (Pmax) of apical tips was calculated from continuous oxygen consumption and evolution measured in a closed system fitted with a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK). Dark respiration monitored by oxygen consumption (Table 1) was added to net photosynthesis to give Pmax. The system was calibrated with oxygen-depleted and oxygen-saturated ASW and the oxygen concentration calculated according to the temperature (15°C) and salinity of the medium (Green & Carritt, 1967). Individual tips were dark adapted for 30 min before transfer to the darkened incubation chamber, which contained 10 ml of one of the Cu2+-enriched or control medium, and oxygen consumption (dark respiration) was measured. Subsequently, an actinic light source, Red LEDs (Hansatech) was switched on at 365 m−2 s−1 and oxygen evolution was measured upon achieving steady-state photosynthesis after approx. 5 min Average oxygen consumption and evolution rates (mol O2 g−1 f. wt min−1) were calculated for a 5–10 min measuring period.
Table 1. Dark respiration rates of Fucus from three populations during exposure to 844 nm Cu2+
Populations (mol O2 g−1 f. wt min−1)
Data are means ± 1 SE, n = 5.
0.16 ± 0.04
0.08 ± 0.03
0.10 ± 0.02
0.17 ± 0.04
0.08 ± 0.02
0.14 ± 0.02
0.15 ± 0.03
0.09 ± 0.02
0.10 ± 0.03
0.13 ± 0.05
0.08 ± 0.08
0.12 ± 0.03
0.20 ± 0.17
0.17 ± 0.06
0.10 ± 0.03
Statistical tests were carried out using the software package statgraphics plus 5.0. Before all parametric tests, the data was tested for homogeneity of variance and normality (Sokal & Rohlf, 1995). Data was subjected to two-factor analysis of variance and differences between individual means were determined by post hoc multiple range tests (Tukey test) at P < 0.05 for this procedure. Errors are displayed graphically as ± 1 SE.
Effects of Cu2+ on algae growth
Relative growth rate of adults Figure 2a shows the effect of 23 d of exposure to Cu2+ on RGRs of vegetative frond tips of adult Fucus. All three populations exhibited a reduction in RGRs as a function of copper exposure but the responses were not the same, as indicated by a significant interaction between populations and treatments (P < 0.001). Bantham and Wembury material responded in a similar way with RGRs significantly inhibited at [Cu2+]ext of 211 nm and above and with negative RGRs when [Cu2+]ext was increased to 844 nm. In comparison, RGRs of Restronguet fronds were unaffected by [Cu2+]ext of 211 nm and while there was a reduction in growth at higher [Cu2+]ext this was not as great as that of the Bantham and Wembury material; the average RGR of Restronguet material at [Cu2+]ext of 844 nm was 1.9% d−1.
Relative growth rate of embryos The pattern of the response to Cu2+ exposure of Fucus embryos obtained from the different locations was similar to that of the respective parent plants (Fig. 2b). The significant population–treatment interaction (P < 0.001) indicates differences in the responses of populations to Cu2+. The RGRs of both Bantham and Wembury embryos were significantly reduced on exposure to [Cu2+]ext of 211 nm and 844 nm. Embryos from Restronguet displayed a significantly greater degree of resistance to all elevated [Cu2+]ext compared with those from Bantham and Wembury, despite a significant reduction in growth at a concentration of 844 nm.
Total copper content of adults The total copper content of apical tips (whole-tissue average) was determined after 23 d of exposure to the various Cu2+ treatments. Material from all three sites accumulated copper in a concentration dependent-manner (Fig. 3), although the pattern of accumulation differed in the three populations (population–treatment interaction, P < 0.001). Bantham and Wembury fronds showed significantly higher CuT contents on exposure to increasing [Cu2+]ext compared with Restronguet plants. At a [Cu2+]ext of 844 nm the CuT content of Restronguet apical tips was approximately 50% that of Bantham and Wembury material.
Effects of Cu2+ on photosynthesis
Chlorophyll fluorescence parameters The effects of Cu2+ on chlorophyll fluorescence parameters of apical tips after 12 d of exposure are presented in Fig. 4. For all parameters measured there were significant main effects and interaction between population and copper treatment. The maximum efficiency of PSII (Fv/Fm) of controls from the three populations was similar (Fig. 4a), with values slightly lower than those recorded at the field sites (approx. 0.8, not shown). Whereas the Fv/Fm of Bantham and Wembury material significantly increased on exposure to [Cu2+]ext of 211 nm and above, increased Fv/Fm values of Restronguet occurred only on exposure to 844 nm. Shorter periods (2 d and 6 d) of exposure to [Cu2+]ext had no significant affect on Fv/Fm for any of the populations (data not shown).
The increase in Fv/Fm of Bantham and Wembury fronds coincided with a significant decrease in Fo (Fig. 4b) and to a lesser extent Fm (Fig. 4c). By contrast, there was no significant effect of Cu2+ on either Fo or Fm of Restronguet fronds (Fig. 4b,c).
The light-saturated quantum yield of photosystem II (ΦPSII) of apical tips exposed to Cu2+ is shown in Fig. 5a. For Bantham and Wembury material the values fluctuated but there were no significant differences between controls and [Cu2+]ext up to 844 nm Cu2+, whereas for Restronguet samples there was no effect of [Cu2+]ext on ΦPSII up to 422 nm, while exposure to 844 nm significantly reduced ΦPSII by 21% compared with controls.
The effects of 12 d Cu2+ exposure on NPQ for the three population samples are shown in Fig. 5b. The NPQ of Restronguet controls was significantly lower than both Bantham and Wembury material. Exposure of Restronguet fronds to Cu2+ produced a significant increase in NPQ at all [Cu2+]ext up to 844 nm, whereas for Bantham and Wembury samples there was a significant increase in NPQ only up to 422 nm. At 844 nm Cu2+. Restronguet fronds had significantly higher NPQ than either Bantham or Wembury samples. At all [Cu2+]ext the NPQ of Wembury fronds was significantly higher than that of Bantham material.
This trend of interpopulation differences in NPQ is also apparent after 23 d exposure to Cu2+ (Fig. 5c). Restronguet material again exhibited a large and significant increase in NPQ when exposed to [Cu2+]ext up to 844 nm, while that of Bantham and Wembury material initially increased up to 211 nm, but thereafter significantly declined at higher [Cu2+]ext.
Oxygen evolution and consumption
Figure 6 shows the effect of Cu2+ on gross photosynthesis (Pmax) expressed by oxygen evolution of adult frond tips from the three populations. There were inhibitory effects on Pmax of Bantham and Wembury fronds exposed to [Cu2+]ext above 211 nm. Oxygen evolution of Restronguet fronds was generally higher at all [Cu2+]ext than that of Bantham and Wembury fronds, although this population showed the same general trend towards inhibition with increasing [Cu2+]ext.
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.
We are grateful to Dr Trevor Burridge (Victoria University, Australia) for critical comments on the final version of the paper. We also thank Dr Maria Donkin (Plymouth University) for commenting on an early draft of the manuscript as well as the interpretation of the fluorescence data. H. D. Nielsen is grateful for receipt of a Plymouth University postgraduate studentship. S. M. Coelho is supported by a scholarship from FCT, Portugal. Work in C. Brownlees laboratory is supported by BBSRC and NERC.