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Keywords:

  • Fucus;
  • cell polarity;
  • copper;
  • signalling;
  • stress response;
  • toxicity mechanism;
  • zygote

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Elevated concentrations of Cu2+ can have inhibitory effects on early development in plants and algae by targeting specific cellular processes. In the present study the effects of elevated Cu2+ on developmental processes in embryos of the brown algae Fucus serratus (Phaeophyceae) were investigated. Elevated Cu2+ was shown to inhibit fixation of the zygotic polar axis but not its formation. Actin localization was unaffected by elevated Cu2+ but polarized secretion, which occurs downstream, was inhibited. Significant differences in tolerance to Cu2+ were observed for polarization and rhizoid elongation of embryos derived from adults from Cu2+-contaminated and uncontaminated locations. Moderate Cu2+ exposure inhibited the generation of cytosolic Ca2+ signals in response to hypo-osmotic shocks. In contrast, cytosolic Ca2+ was elevated by treatments with high [Cu2+] and this coincided with production of reactive oxygen species. The results indicate that direct effects on signalling processes involved in polarization and growth may in part explain complex, concentration-dependent effects of Cu2+ on early development.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Cu2+ is an essential nutrient required for metabolic processes in all eukaryotes. but can reach toxic levels in both terrestrial and aquatic environments, particularly in copper-contaminated soils adjacent to mines and smelters, and in estuaries and coastal systems that receive mine drainage water (Apte et al. 1990; Bryan & Langston 1992; Widerlund 1996; Ng et al. 1996; DeGregori et al. 1996; Austen & Somerfield 1997; Marsden & DeWreede 2000; Vasquez et al. 2000; Grout & Levrings 2001; Eriksen et al. 2001; Stauber et al. 2001). Although certain species of higher plants such as Salix spp., Caluna vulgaris and Silene vulgaris have been shown to have the capacity to acquire a significant degree of metal tolerance (Landberg & Greger 1996; Monni et al. 2000; van Hoof et al. 2001), the effects of Cu2+ at different developmental stages in plants has not been studied in detail. It is known, however, that the early life history stages of marine invertebrates and macroalgae may be particularly sensitive to elevated total copper concentrations [CuT] and that different developmental stages may be affected differently (Andersson & Kautsky 1996; Bidwell, Wheeler & Burridge 1998; Botton, Johnson & Helleby 1998; King & Riddle 2001).

Certain macroalgal species including Enteromorpha spp. and Fucus spp. are able to grow in copper-contaminated seawater (Seeliger & Cordazzo 1982 ; Bryan & Gibbs 1983; Correa et al. 1996; Marsden & DeWreede 2000). Fucoid algae in particular provide good potential model systems for studying metal tolerance and toxicity mechanisms because the physiology of their early development is well studied and zygotes and embryos are easy to obtain and culture.

In oceanic surface waters the total copper concentration [CuT] is governed largely by biological activity and is maintained at pm–nm concentrations (Bruland 1980; Coale & Bruland 1990, Sunda & Huntsman 1995). Within the Fal Estuary system in the United Kingdom, Restronguet Creek (Fig. 1) is particularly contaminated and contains an average of about 0.2 µm[CuT] which is 20-fold higher than that found in uncontaminated estuaries (Bryan & Langston 1992; The Environment Agency, UK, unpublished results). Adult Fucus serratus specimens from uncontaminated sites may tolerate up to 0.2 µm CuT though higher [CuT] may reduce photosynthesis and growth. In contrast, F. serratus growing in a copper-contaminated site such as Restronguet Creek can tolerate exposure to up to 1 µm CuT (Strömgren 1980; Bryan & Gibbs 1983; Nielsen et al. unpublished results).

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Figure 1. Map of the sampling area showing the gird references (OS Grid) of the sampling sites.

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In the Fucus zygote, developmental events during the first few hours after fertilization (AF) and prior to germination may be more sensitive to Cu2+ than embryo and adult growth. The addition of 0.04 µm CuT at 6‰ salinity to Fucus vesiculosus gametes at the time of fertilization reduced germination success by about 50% (Andersson & Kautsky 1996). When added after polarization there was no effect of 0.2–0.6 µm CuT on Fucus rhizoid elongation which was significantly reduced at higher [CuT] and became irreversibly arrested when [CuT] was increased to 2 µm (Andersson & Kautsky 1996; Gledhill et al. 1999; Bond et al. 1999). Similarly, there were inhibitory effects of elevated concentrations of Cu2+ on Macrocystis gametophyte germ tube elongation (Anderson et al. 1990; Bidwell et al. 1998; Burridge, Karistianos & Bidwell 1999).

During early Fucus zygote development a polar growth axis is first formed and then fixed (Berger & Brownlee 1994). This is critically dependent on a series of cytological events involving filamentous actin localization (Alessa & Kropf 1999; Pu, Wozniak & Robinson 2000) and localized secretion of the Golgi-derived sulphated polysaccharide, fucoidin into the cell wall (Shaw & Quatrano 1996). Polarized signals underlying these localizations include localized Ca2+ elevation at the rhizoid pole that is maintained, at least in part, by ion channel mediated influx of Ca2+ across the plasma membrane (Taylor, Roberts & Brownlee 1992; Taylor & Brownlee 1993; Berger & Brownlee 1993; Roberts, Berger & Brownlee 1993; Taylor et al. 1996; Taylor, Manison & Brownlee 1997; Pu & Robinson 1998). The present study sets out to determine the susceptibility of different stages of Fucus zygote development to Cu2+ exposure and to examine mechanisms of toxicity and tolerance. This was achieved by: (1) examining effects of Cu2+ on formation and fixation of the polar axis and rhizoid elongation of embryos obtained from populations from copper-contaminated and uncontaminated locations; (2) pinpointing early cytological events which may be targeted by Cu2+ and (3) assessing the effect of Cu2+ on intracellular Ca2+ signalling and production of reactive oxygen species (ROS).

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Individuals of Fucus serratus bearing mature fronds were collected from three populations near Plymouth, UK (Fig. 1). Adult plants collected from Restronguet Creek were subject to anthropogenic Cu2+ pollution, whereas those collected from Bantham Quay in the Avon Estuary and Wembury Beach were exposed to natural background estuarine or coastal concentrations, respectively (Bryan & Langston 1992; The Environment Agency, UK, unpublished results).

Mature receptacles were cut from separate male and female fronds, rinsed in seawater, blotted dry and stored wrapped in paper towels in the dark at 3–5 °C for up to 10 d. Gamete release was stimulated by thoroughly rinsing the receptacles in tap water followed by exposure to natural sunlight for 15 min. The receptacles were transferred to UV-treated and filtered (0.45 µm cellulose nitrate membrane) natural seawater (FSW) and male and female gametes were released separately within 1 h. Mixing sperm and eggs under fluorescent white light at 16 °C for 30 min induced fertilization. The resulting zygotes were filtered through a 100 µm nylon mesh into FSW. The zygotes were sown onto small Petri dishes (2.5 cm diameter) with coverslip bases (> 200 per dish) and incubated at 16 °C in continuous unidirectional fluorescent white light (50 µmoles m−2 s−1). After adhesion to the coverslips, the zygotes were transferred to artificial seawater culture medium (ASW) modified from the original recipe according to Morel et al. (1979) and Gledhill et al. (1999). There was no difference in zygote growth performance in ASW and FSW (data not shown). During experiments, ASW was enriched with CuSO4·H2O to yield [CuT] ranging between 0 and 20 µm. Concentrations of free Cu2+ in Cu2+-supplemented ASW were calculated by applying the total concentration of the ASW constituents to the chemical equilibrium-modelling program minequl+ 3.01 (Westall, Zachary & Morel 1976; Morel et al. 1979; Gledhill et al. 1999).

Effect of Cu2+ on rhizoid elongation in different populations

To determine the Cu2+-tolerance level of Fucus embryos from the three populations (see above) zygotes were allowed to develop in Cu2+-free ASW until 18 h after fertilization (AF) when germination was initiated, and subsequently either transferred to ASW containing 211 nm Cu2+ or retained in Cu2+-free medium and grown for a total of 8 d. Rhizoids maintained a constant rate of elongation for up to 8 d in Cu2+-free medium, presumably due to the availability of maternally supplied Cu2+ (data not shown). Fucus embryos release metal-complexing ligands when exposed to metals, which bring about a decrease in the external Cu2+ concentration ([Cu2+]ext) (Gledhill et al. 1999). To compensate for ligand release from the growing embryos the medium was changed every day. Rhizoid length defined as the distance from the rhizoid/thallus-dividing wall to the rhizoid tip was recorded digitally from bright field images and was measured using analytical software (lucida; Kinetic Imaging, Liverpool, UK).

Timing of axis formation and fixation

In response to unidirectional light, Fucus zygotes first form a labile polar axis, which is subsequently irreversible fixed and culminates in germination of a rhizoid away from the light direction. To determine the period of axis formation and fixation, synchronously developing batches of zygotes were grown in ASW in unidirectional light (L1). At 1 h intervals from 2 to 14 h AF, batches of zygotes were either transferred to darkness to determine the proportion of zygotes that had formed an axis or to unidirectional light from the opposite direction of L1 (L2) to determine the proportion of zygotes that had fixed an axis in response to L1. Zygotes that had formed or fixed an axis prior to transfer to either darkness or L2 would germinate away from L1. In contrast, zygotes that had not formed an axis upon transfer to darkness would germinate in a random direction rather than away from L1, whereas zygotes that had not fixed an axis upon transfer to L2 would germinate away from L2 rather than L1. The proportion of zygotes that had formed or fixed an axis was scored upon germination at 24 h AF and calculated as: (number of zygotes germinating in the hemisphere away from L1)/(total number of germinating zygotes).

Effects of Cu2+ on axis formation and fixation

Zygotes were incubated in Cu2+-free ASW in L1 and were transferred, while still in L1, to ASW containing concentrations of Cu2+ ranging from 0 to 2110 nm at the beginning of either the phase of axis formation or fixation. Zygotes were then transferred to Cu2+-free ASW either in darkness to determine the proportion that had formed their axes, or to L2 to determine the proportion that had fixed their axes. Upon germination, at 24 h AF, the proportions of zygotes that had formed or fixed their axes during Cu2+ exposure were scored.

F-actin localization

Polarizing Fucus zygotes localize F-actin at the rhizoid pole during axis fixation (7–13 h AF; Kropf 1997). Zygotes were incubated in ASW either with or without 2110 nm free Cu2+ in L1 during axis fixation (7–13 h AF). Following this, F-actin distribution was visualized using the F-actin-specific fluorescent probe Texas Red phalloidin (Molecular Probes, Eugene, OR, USA), and confocal microscopy (Henry, Jordan & Kropf 1996). Zygotes were transferred to 1 mL formaldehyde fixative (0.1 mm maleimidobenzoyl-N-hydroxysuccinimide ester, 3.8% formaldehyde, 0.2% dimethylsulfoxide in ASW) for 30 min then stained for 30 min in Texas Red phalloidin solution (1.3 µm Texas Red phalloidin in ASW + 0.2 m sorbitol). Stained zygotes were washed three times for 5 min in ASW + 0.2 m sorbitol. Fluorescent images of F-actin distribution were obtained using a MRC 1024 confocal laser scanning microscope (Bio-Rad, Hemel Hempstead, UK). An argon/krypton laser excited the Texas Red at 568 nm and emission was recorded at 605 nm with a 10-nm bandwidth 605 nm emission filter. The proportion of zygotes which had localized F-actin was scored.

Secretion of fucoidin

During axis fixation (7–13 h AF) zygotes were transferred to ASW containing concentrations of Cu2+ ranging from 0 to 2110 nm. Secretion of fucoidin into the cell wall was then visualized by staining with Toluidine Blue-O (TB-O) (Sigma Chemicals, Poole, Dorset, UK). Zygotes were transferred to the TB-O sulution (0.1% TB-O in ASW, pH 1.5 with HCl) for 15 min and washed three times for 5 min in 99% ethanol (Shaw & Quatrano 1996). Stained zygotes were mounted in ASW and the proportion of zygotes, which had secreted fucoidin into the cell wall was scored under a light microscope.

Effects of Cu2+ on cell division

Zygotes were transferred to ASW containing concentrations of Cu2+ varying from 0 to 844 nm by 2 h AF. At 40 h AF the embryo partition membranes were labelled by immersing embryos for 10 min in the fluorescent membrane-specific dye N-(3-triethyl-ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 1–43) (Molecular Probes). Images of more than 25 embryos for each treatment were recorded with a confocal microscope (see above) at 488 nm excitation and 530 nm emission.

Cytosolic Ca2+

Cytosolic Ca2+ was monitored ratiometrically by monitoring the fluorescence of Calcium Green, a Ca2+ -sensitive fluorescent dye and Texas Red, a Ca2+-insensitive indicator. Germinated rhizoids of zygotes at the two-cell stage were pressure microinjected with an artificial intracellular solution comprising: 5 mm Hepes; 200 mm KCl; 550 mm, mannitol; 1 mm Calcium Green 10 000 dextran (Molecular Probes); 1 mm Texas Red 10 000 dextran (Molecular Probes), pH 7.4 as described previously (Berger & Brownlee 1993; Coelho et al. 2002). Zygotes were transferred to ASW containing 0.6 m sorbitol and viewed on an inverted microscope (Nikon, Tokyo, Japan). Impaling of an embryo 20–30 µm from the rhizoid apex was carried out under white light at a holding pressure of 20 kPa established in the pipette by a Pico-injector LPI-100 (Medical Systems, Greenvale, NY, USA). Dye was injected at a pressure of 220 kPa and dye fluorescence was monitored with 480 nm excitation and 530 nm emission. In vitro calibration of the dye concentration was obtained from confocal images of droplets of intracellular solution containing Texas Red concentrations varying from 0 to 250 µm. The microinjection procedure produced an intracellular dye concentration of about 50 µm (Coelho et al. 2002). In the present study this was confirmed by comparing the intracellular fluorescence of Texas Red with the fluorescence of solutions of known Texas Red concentrations. Confocal images of Calcium Green and Texas Red were acquired simultaneously. An argon-krypton laser excited Calcium Green at 488 nm and Texas Red at 568 nm and emission wavelengths were 532 nm for Calcium Green and 605 nm for Texas Red. The Calcium Green images were ratioed against the corresponding Texas Red images using lucida software (Kinetic Imaging). In vitro calibration of cytosolic [Ca2+] was carried out from Calcium Green/Texas Red confocal ratio images of droplets of Ca2+ buffer solutions containing Ca2+ concentrations between 0.01 and 10 µm (Tsien & Rink 1980) and 50 µm Calcium Green/Texas Red.

ROS production

Intracellular ROS production was monitored as oxidation of 5- (and 6-) chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFH2-DA; Molecular Probes; Coelho et al. 2002). Germinated zygotes were transferred to ASW containing 10 µm CM-DCFH2-DA and 1% dimethyl sulphoxide (DMSO) for 20 min. To avoid wash out of dye from the cytoplasm, dye was retained in the experimental solutions (Coelho et al. 2002). Confocal images were obtained after excitation at 488 nm and emission at 522 nm. ROS production was expressed relative to the resting level.

Effects of Cu2+ on osmoregulation

Eighteen-hour-old embryos were transferred to ASW containing concentrations of Cu2+ varying from 0 to 2110 nm for 6 h. The incubation chambers were drained and embryos were flushed with 5 mL ASW diluted with distilled water to either 50 or 25% ASW. The proportion of embryos which were able to osmoregulate during hypo-osmotic exposure was then scored and calculated as: 1 − (number of embryos bursting during hypo-osmotic treatment/total number of embryos).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Effect of Cu2+ on rhizoid elongation

Figure 2 shows the effect of Cu2+ on rhizoid length after 8 d of culture. Fucus embryos from algae collected from the copper-exposed Restronguet Creek location had a significantly greater Cu2+ tolerance than those from algae occupying unexposed habitats at Wembury Beach and Bantham Quay locations, which had similar low Cu2+ tolerance. When exposed to 0 and 211 nm Cu2+ there was no significant difference in rhizoid length between Bantham and Wembury embryos (P > 0.05; Fig. 2a). Rhizoid length of Bantham and Wembury embryos exposed to 211 nm Cu2+ was reduced significantly compared with control embryos (P < 0.05; Fig. 2a). In contrast, there appeared to be some Cu2+ requirement for rhizoid elongation in embryos from algae collected from the Cu-contaminated Restronguet Creek location. Thus, rhizoids of Restronguet embryos cultured in Cu2+-free control medium were significantly shorter than rhizoids of Bantham and Wembury control embryos (P < 0.05). However, when cultured in 211 nm Cu2+ rhizoid length of Restronguet embryos increased significantly in comparison with Restronguet control embryos (P < 0.05; Fig. 2a).

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Figure 2. (a) Rhizoid length, after 8 d of exposure to Cu2+, of Fucus embryos from populations occupying clean sites at Wembury Beach (unshaded bars) and Bantham Quay (shaded bars) and the Cu2+-contaminated site at Restronguet Creek (solid bars). (b, c) Examples of rhizoid elongation after 8 d of exposure to Cu2+-free control medium and medium containing 211 nm Cu2+ for Fucus embryos from Wembury Beach (b) and Restronguet Creek (c).

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Cu2+ disrupts axis fixation but not formation

Figure 3a shows the proportion of Wembury zygotes in ASW that had formed or fixed their axes at different times AF as expressed by the proportion of zygotes which polarized in response to a unidirectional light source (L1). Fifty percent polarization before 3 h AF denotes lack of axis formation; 100% polarization at 7 h AF denotes complete axis formation. Axis fixation was complete by 13 h AF. There were specific inhibitory effects of Cu2+ on different stages of axis establishment. Notably, axis formation in Wembury zygotes was unaffected by [Cu2+]ext up to 2110 nm with at least 93% of zygotes forming a polar axis (Fig. 3b & c). However, axis fixation in the same zygotes was significantly inhibited by [Cu2+]ext at 211 nm and above, and an increase in [Cu2+]ext from 84.4 to 2110 nm resulted in a decrease in axis fixation (P < 0.05; Fig. 3b & d). In Restronguet zygotes there appeared to be some Cu2+ requirement for axis fixation such that [Cu2+]ext up to 211 nm increased the number of zygotes fixing their axis (P < 0.05; Fig. 3b). Further increase in [Cu2+]ext above 211 nm resulted in a decrease in axis fixation compared with untreated controls (P < 0.05).

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Figure 3. (a) Polarization in batches of Fucus zygotes, exposed to unidirectional light (L1) and transferred to dark to determine axis formation (unshaded bars) or to reversed unidirectional light (L2) to determine axis fixation (solid bars) at various times AF. The proportion of zygotes that had formed or fixed an axis in response to L1 was scored upon germination. Axis formation occurred between 3 and 7 h AF and axis fixation was apparent between 7 and 13 h AF. (b) Proportion of polarization in batches of Wembury (uncontaminated site) zygotes exposed to various [Cu2+]ext during axis formation (○) and fixation (□), and in batches of Restronguet (contaminated site) zygotes during axis fixation (▪). Means ± 1 SD, n = 4. (c) Examples of zygotes forming their axis between 3 and 7 h AF in response to L1 during exposure to Cu2+-free control medium (left) or medium containing 2110 nm Cu2+ (right). Images show zygotes germinating in darkness following L1 treatment. Although some of the Cu2+ exposed zygotes in this example have orientated their axis at a right angle to L1, there was no significant overall difference between treatments. (d) Inhibition of axis fixation in Wembury control zygotes and zygotes exposed to 2110 nm Cu2+ between 7 and 13 h AF.

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Effects of Cu2+ on F-actin localization and secretion of fucoidin

Figure 4a shows the effect of Cu2+ on F-actin localization in Wembury zygotes by 13 h AF. Eighty percent of zygotes (n = 56) had localized F-actin at the rhizoid pole after exposure to 2110 nm Cu2+ during axis fixation (7–13 h AF). Although this is slightly lower than the 97% of control zygotes (n = 32) which localized F-actin during axis fixation, it is considerably higher than the proportion of zygotes which irreversibly fixed their axis during exposure to 2110 nm Cu2+ (see Fig. 3). The disruption of F-actin localization alone can not therefore account for the inhibitory effect of Cu2+ on axis fixation. Figure 4b shows the effect of Cu2+ on polarized secretion of fucoidin in Wembury zygotes monitored 13 h AF. At [Cu2+]ext up to 84.4 nm there was no effect on secretion of fucoidin (P > 0.05), whereas an increase in [Cu2+]ext from 84.4 nm to 422 nm strongly inhibited localized secretion. The inhibitory effect of Cu2+ on localized secretion occurred within the same concentration range as that which inhibited axis fixation. Figure 4c shows examples of secretion of fucoidin by 13 h AF in control zygotes as visualized by Toluidine Blue-O (TB-O) staining (left) and the inhibitory effect on localized secretion of fucoidin of exposure to 211 nm Cu2+ in TB-O-stained zygotes (right). Taken together these results indicate that the effect of Cu2+ on axis fixation reflects either direct inhibition of polarized secretion or a process that occurs downstream of F-actin localization leading to polarized secretion.

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Figure 4. (a) Examples of F-actin distribution in Wembury zygotes 13 h AF. F-actin localization at the rhizoid pole occurred in 97% of control zygotes not exposed to Cu2+ during axis fixation (n = 56) (i). 80% of zygotes exposed to 2110 nm Cu2+ during axis fixation localized F-actin at the rhizoid pole (ii), whereas F-actin remained uniformly distributed in 20% (iii) (n = 32). (b) Proportion of Wembury zygotes secreting fucoidin at the rhizoid pole by 13 h AF when exposed to various [Cu2+]ext during axis fixation. Means ± 1 SD, n = 3. (c) Example of fucoidin secretion visualized by Toluidine Blue-O staining of control zygotes, not exposed to Cu2+ during axis fixation (left) and inhibition of polarized secretion in zygotes exposed to 211 nm Cu2+ during axis fixation (right).

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Effects of Cu2+ on cell division

In embryos cultured in various [Cu2+]ext immediately after fertilization, there was a positive effect of low to moderate concentrations, on the number of cells per embryo whereas high [Cu2+]ext inhibited cell division (Fig. 5a); 42.2 nm Cu2+ increased the number of cells per embryo significantly, whereas higher [Cu2+]ext progressively decreased the number of cells per embryo. Whereas the the cell division pattern was disrupted in only 5.8% of embryos after 40 h of culture in 42.2 nm Cu2+, there was an abnormal cell division pattern in 52% of embryos exposed to 2110 nm Cu2+ (Fig. 5b).

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Figure 5. (a) Number of cells of Wembury embryos exposed to various [Cu2+]ext from 2 h AF to 24 h AF (○) and 40 h AF (•). Means ± 1 SD, n = 3. (b) Examples of cell division patterns by 40 h AF. Normal cell division patterns were observed in 94% of embryos (> 75) exposed to 42.2 nm Cu2+ (left) whereas abnormal cell division patterns were observed in 52% of embryos (> 75) exposed to 2110 nm Cu2+ (right).

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Cu2+ disrupts Ca2+ signalling in the Fucus rhizoid

We compared short-term and chronic exposure to moderate [Cu2+]ext (422 nm) on Ca2+ signalling in the Fucus rhizoid. In the absence of Cu2+, osmotic shock (transfer from ASW to 80% ASW) produced within 20 s an abrupt 10-fold increase in the cytosolic Ca2+ concentration in the rhizoid apex ([Ca2+]cyt), from approximately 0.1–1 µm, lasting at least 1 min (Fig. 6a & e). The Ca2+ elevation was largely restricted to an area near the cell membrane, extending a few micrometres into the cytoplasm. This is consistent with previous reports of the effect of small hypo-osmotic shocks on [Ca2+]cyt (Goddard et al. 2000). The ability of embryos to increase apical [Ca2+]cyt abruptly in response to hypo-osmotic conditions was inhibited by moderate [Cu2+]ext both during short-term and chronic exposure (Fig. 6e). In four out of six embryos pretreated with 422 nm Cu2+ for 5 min there was no elevation in [Ca2+]cyt during exposure to hypo-osmotic conditions (Fig. 6b). Only minor increases in [Ca2+]cyt, much less than in the control embryos, were observed in the two remaining embryos (e.g. Fig. 6c). Figure 6d shows an example of [Ca2+]cyt during an 80% hypo-osmotic shock in embryos pretreated with 422 nm Cu2+ for 3 h. In five out of seven embryos no [Ca2+]cyt elevation occurred in response to hypo-osmotic exposure. In the remaining two embryos a response, similar to the one shown in Fig. 6c, was observed. Figure 6f–h show examples of [Ca2+]cyt during a 80% hypo-osmotic shock in the subapical region of control embryos and embryos pretreated with 422 nm Cu2+ for 5 min or 3 h. Sub-apical [Ca2+]cyt in these embryos was not affected significantly by hypo-osmotic exposure.

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Figure 6. (a–d) Calcium green/Texas red ratio images showing the effect of Cu2+ on changes in [Ca2+]cyt in the rhizoid apex (apical 0–5 µm) of Wembury embryo rhizoids 24 h AF during an 80% hypo-osmotic shock. (a) A control embryo not exposed to Cu2+. (b, c) Embryos subject to short-term (5 min) exposure to 422 nm Cu2+ prior to hypo-osmotic shock. (d) An embryo subject to chronic (3 h) exposure to 422 nm Cu2+ prior to the hypo-osmotic shock. (f–h) [Ca2+]cyt in subapical regions (20–25 µm from apex) of Wembury embryo rhizoids 24 h AF in response to an 80% hypo-osmotic shock. (f) A control embryo not exposed to Cu2+. (g) An embryo subject to short-term (5 min) exposure to 422 nm Cu2+ prior to the hypo-osmotic shock (h) An embryo subject to long-term (3 h) exposure to 422 nm Cu2+ prior to the hypo-osmotic shock. There is 5 s between images and time zero is taken as the time of application of the hypo-osmotic treatment. (e) Average changes in apical [Ca2+]cyt during exposure to an 80% hypo-osmotic shock in embryos subject to short-term, n = 6 (▪) and long-term, n = 7 (▴) exposure to 422 nm Cu2+ and in control embryos, n = 3 (•).

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Acute exposure to 2110 nm Cu2+ caused an approximately four-fold increase in Ca2+ within 30 s of exposure (Fig. 7). In the cell shown [Ca2+]cyt increased from 0.15 to 0.6 µm throughout the rhizoid cell, and remained at this level for at least 2 min. Increase in [Ca2+]cyt during acute exposure to high [Cu2+]ext was observed in all embryos (n = 5) exposed to various high [Cu2+]ext ranging from 0.844 to 8.44 µm.

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Figure 7. (a) Calcium Green/Texas Red ratio images of the [Ca2+]cyt observed during 1 h in a Wembury Fucus rhizoid 24 h AF subject to acute exposure to 2110 nm Cu2+. Time zero is taken as the time of application of Cu2+. (b) Changes in average [Ca2+]cyt (means ± SE, n = 5) in the Fucus rhizoid.

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Effect of Cu2+ on ROS production

Figure 8a shows an example of intracellular ROS production in a Fucus rhizoid exposed to 2110 nm Cu2+. Cu2+ stimulates an immediate but steady increase in ROS production in the subapical rhizoid region (Fig. 8b).

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Figure 8. (a) Confocal images of intracellular ROS production in a Fucus rhizoid exposed to 2110 nm Cu2+. (b) Average (means ± SE, n = 5) increase in intracellular ROS during 160 s in the Fucus rhizoid subject to exposure to 2110 nm Cu2+. Time zero is taken as the time of application of Cu2+.

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Effects of Cu2+ on osmoregulation

The ability of zygotes to osmoregulate in response to a hypo-osmotic shock was monitored by counting the numbers of embryos that burst in response to transfer ASW to 50 or 25% ASW. Osmoregulation in embryos cultured in various [Cu2+]ext subsequent to axis fixation was either unaffected or improved (Fig. 9). The proportion of embryos able to osmoregulate during exposure to a 50% hypo-osmotic shock was unaffected by [Cu2+] up to 844 nm. Further increase in [Cu2+]ext to 2110 nm significantly increased the proportion of embryos which did not burst upon exposure to 50% ASW. A similar effect of Cu2+ on osmoregulation was found for embryos exposed to 25% hypo-osmotic shock.

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Figure 9. Osmoregulation in batches of Wembury zygotes, measured as the proportion of zygotes that could withstand hypo-osmotic treatment without bursting, in embryos exposed to various [Cu2+] from 18 h to 24 h AF and subsequently given a 50% hypo-osmotic shock (○) or a 25% hypo-osmotic shock (•). Means ± 1 SD, n = 3.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The results presented here show that elevated concentrations of Cu2+ can have inhibitory effects on specific developmental processes. The effects of Cu2+ on early development and growth have been described for a number of species, including brown algae (Andersson & Kautsky 1996; Botton et al. 1998; King & Riddle 2001) and effects on rhizoid growth in Fucus and Eclonia are well documented (Bidwell et al. 1998; Bond et al. 1999; Gledhill et al. 1999). Here we show further that Cu2+ has specific inhibitory effects on fixation but not formation of the zygotic polar axis in Fucus. Axis fixation is required for correct orientation of the first cell division plane ( Allen & Kropf 1992; Corellou et al. 2000) and the observed abnormal cell divisions in zygotes exposed to Cu2+ are consistent with disruption of this process.

Our results indicate that the effect of Cu2+ on axis fixation is related to the disruption of a process occurring downstream of F-actin localization and which is required for polarized secretion. F-actin localization at the rhizoid pole is essential for early polarization in fucoid zygotes (Bouget et al. 1998; Alessa & Kropf 1999; Pu et al. 2000). We show here that F-actin localization is unaffected by exposure of zygotes to Cu2+ concentrations that had pronounced effects on axis fixation and rhizoid growth. This is consistent with the observation that various divalent metal ions including Cu2+ were ineffective in depolymerizing the F-actin cytoskeleton of vascular smooth muscle cells (Wang, Chin & Templeton 1996). In addition to actin localization, polarized secretion of Golgi-derived sulphated polysaccharide (fucoidin) into the cell wall at the rhizoid pole is also essential for polarization and occurs subsequent to, or simultaneous with, F-actin localization (Wagner, Brian & Quatrano 1992; Shaw & Quatrano 1996). Secretory vesicles containing fucoidin may be guided to the rhizoid pole via F-actin (Goodner & Quatrano 1993; Goode, Drubin & Barnes 2000) and it has been proposed that localization of fucoidin at the rhizoid pole may provide anchorage sites for axis stabilization complexes involving the actin cytoskeleton (Goodner & Quatrano 1993). Significantly, we show here that Cu2+ has a significant inhibitory effect on localized secretion of fucoidin. In the Fucus rhizoid and Lilium pollen tube inhibition of elongation by Cu2+ coincided with the accumulation of secretory vesicles within the cell apex (Sawidis & Reiss 1995; Bond et al. 1999).

Cytosolic Ca2+ is thought to regulate the events underlying polarization in fucoid zygotes (Speksnijder et al. 1989; Berger & Brownlee 1993; Roberts et al. 1993; Pu & Robinson 1998) as well as apical growth in several polarized plant and algal cell types such as algal rhizoids, pollen tubes and root hairs (Hepler, Vidali & Cheung 2001) . Both short- and long-term exposure to moderate [Cu2+]ext (422 nm) attenuated the growth-related Ca2+ gradient and the elevation of Ca2+ in the rhizoid apex in response to hypo-osmotic shock. In contrast, acute exposure to higher [Cu2+]ext (2.11–8.44 µm) resulted in a global cellular [Ca2+] increase. The growth-related apical Ca2+ elevation is initiated at the rhizoid pole of the Fucus zygote during polarization (Berger & Brownlee 1993; Roberts et al. 1993; Pu & Robinson 1998) and is required for rhizoid formation and elongation (Speksnijder et al. 1989; Taylor et al. 1992). Attenuation of the polar Ca2+ signal by moderate [Cu2+]ext may therefore on its own be sufficient to inhibit axis fixation and rhizoid elongation. Ion channel mediated Ca2+ flux from the external medium into the cytoplasm is required for the growth related apical Ca2+ elevation in Fucus rhizoids (Taylor et al. 1996, 1997). The inhibition of apical Ca2+ elevations by Cu2+ suggests an inhibition of channel-mediated Ca2+ influx as one of the early effects of Cu2+. Such effects on Ca2+–permeable channels have been observed in several other systems (Kasai & Neher 1992; Osipenko, Kiss & Salanki 1992; Klusener, Boheim & Weiler 1997).

The demonstration here that Cu2+ levels that lead to transient cellular Ca2+ elevations also result in ROS production in rhizoid cells is consistent with a primary effect of high [Cu2+] via oxidative reactions although the present data do not allow us to determine whether ROS production is necessary for Ca2+ elevation in response to high Cu2+. Exposure of zygotes to high [Cu2+]ext produced elevation of both Ca2+ and H2O2, both of which have been shown to be involved in several stress responses in higher plants (Bowler & Fluhr 2000). Moreover, we have shown recently that production of ROS occurs upstream of Ca2+ signals in response to osmotic shocks in the Fucus rhizoid (Coelho et al. 2002). Several studies have indicated that exposure to Cu2+ induces the production of ROS in higher plants (Navari-Izzo et al. 1998; Teisseire & Guy 2000 ; Quartacci, Cosi & Navari-Izzo 2001). The sequence of events leading to Ca2+ elevation and ROS production remains to be elucidated.

Our data suggest that the mode of action of Cu2+ on the Fucus rhizoid cell varies with different [Cu2+]. Low [Cu2+]ext leads to direct inhibition of Ca2+ dynamics in the rhizoid apex whereas high Cu2+ leads to Ca2+ elevation, possibly in response to ROS production. The improved ability of Cu2+-treated embryos to withstand osmotic treatments is consistent with increased production of ROS, probably external to the plasma membrane, which may have a strengthening effect on the cell wall (Coelho et al. 2002).

Adult Fucus plants growing in the copper-contaminated environment are known to tolerate higher [CuT] than those growing at uncontaminated locations (Bryan & Gibbs 1983). The higher Cu2+ tolerance of axis fixation and rhizoid elongation of embryos obtained from a copper-exposed population compared with that of embryos from an un-exposed population suggests that the ability of brown algae to develop copper tolerance is an inherited rather than an acquired character. Previous comparative studies of Cu2+ toxicity in kelp gametophyte rhizoid elongation showed no interpopulation differences, which may, however, be related to the unclear contamination history of the populations tested (Anderson et al. 1990). The estuarine Cu2+ environment is very heterogeneous (Turner 1996; Ng et al. 1996) and the concentration range (up to 844 nm Cu2+) used in our experiments is representative of that which may be encountered in contaminated estuaries such as Restronguet Creek within a single day (Bryan & Langston 1992; The Environmental Agency of the UK, unpublished results). Low salinity in estuaries and enclosed waters such as the Baltic Sea may affect the copper speciation (Turner 1996; Ng et al. 1996) and result in enhanced sensitivity of developing Fucus embryos to CuT (Andersson & Kautsky 1996). In their natural environment Fucus zygotes are therefore potentially met with peak concentrations of Cu2+ which may disrupt polarization, hence, affecting further embryo development, survival and recruitment into the reproductive population, leading to selection of individuals with higher Cu2+ tolerance. The basis of copper tolerance mechanisms in Fucus, however, is not well understood although the requirement for low [Cu2+] for rhizoid growth and axis fixation in tolerant embryos, indicates that copper tolerance may relate to increased production of chelators, improved exclusion mechanisms or sequestering. Work is currently in progress to determine the nature of copper tolerance and requirement in exposed populations.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

H.N. is grateful for receipt of a Plymouth University postgraduate studentship. Work in C.B.′s laboratory is supported by BBSRC and NERC.

REFERENCES

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  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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