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The effects of copper on the fine structure of the kelp Laminaria saccharina (L.) Lamour
Marine Environmental Research 19 (1986)205-223
The Effects of Copper on the Fine Structure of the Kelp Laminaria saccharina (L.) Lamour* Boudewijn H. Brinkhuis & Ik K y o C h u n g Marine SciencesResearch Center, State Universityof New York, Stony Brook, New York 11794--5000, USA (Received: 28 February, 1986) A BSTRA CT The effects of copper on the fine structure of Laminaria saccharina were examined by incubating plants in 50, 100 and 500 #g Cu per liter of filtered and uv-treated seawater media. Copper treatment after fertilization in gametophytes yielded sporophytes that showed abnormal growth patterns, haptera-like protuburances, giant cells and abnormal branching patterns in 50 #g liter- t and 10012g liter-t copper additions. Ultrastructural examination of the plants which showed abnormality and chlorosis indicated the presence of denatured chloroplasts with swollen thylakoid membranes, detachment of thylakoid lamellae and diffused matrices.
INTRODUCTION Some temperate coastal ecosystems are based on seaweed primary productivity (Mann, 1972a, b). Bioaccumulated metals in seaweed tissue may be transferred to the detrital food web or directly to grazing organisms such as fish and sea urchins. The process of microbial activity in the sediment may be decreased by the metals accumulated in algal detritus. Finally, the total production of the system may be affected by the decreased microbial activity which recycles nutrients to the water column (Babich & Stotzky, 1985). Although there is still uncertainty about the processes involved in * Marine Sciences Research Center Contribution No. 503. 205 Marine Environ. Res. 0141-l136/86/$03-50 :~ ElsevierApplied SciencePublishers Ltd, England, 1986. Printed in Great Britain
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Boudewijn H. Brinkhuis, Ik Kyo Chung
heavy metal accumulation by brown seaweeds, their ability to concentrate ions of metals from the surrounding seawater has been well documented and makes them useful indicators of marine heavy metal pollution (Bryan, 1983; Bryan & Hummerstone, 1973; Haug et al., 1974; Myklestad et al., 1978). The effects of accumulated metals on the plants themselves, however, have been less studied. One of the most commonly observed effects of heavy metal poisoning is a change in cell size or morphology. This has been observed in a wide variety of organisms. The uncoupling of cell growth and cell division (Davies. 1976) and interruption of cell separation, cytoplasmic changes in color and contents and disruption of chloroplast integrity and dispersion (Thomas et al., 1980), and morphological aberration (Sunda & Guillard, 1976) have been observed in phytoplankton species. Methylmercury absorbed by the aquatic plant, Elodea densa, affected chloroplast integrity (Mortimer & Czuba, 1982), which is very important to normal photosynthetic activity. Mortimer & Czuba proposed that the chloroplast membrane may be much more sensitive than other membranes within the cell. The ultrastructure of mitochondria also was affected by heavy metals. Cadmium treated freshwater green algae showed intra-mitochondrial dense granules and altered cristae structure (Silverberg, 1976). A significant decrease in the number of mitochondria in Diatoma species after gold treatment was based on the morphometric analysis (Sicko-Goad & Stoermer, 1979). Algal response to heavy metal exposure indicated that changes in vacuole volume were responsible for metal toxicity that was attributed to changes in membrane permeability (Sicko-Goad, 1982). Few such studies have been reported for seaweeds. The present study was conducted to delineate the morphological effects of copper on the brown alga, Laminaria saccharina, during early sporophyte stages.
MATERIALS AND METHODS The present study followed the culture methods of Brinkhuis et al. (1983, 1984). An incubation chamber was maintained at 10 + I°C and illuminated by 'very high output' cool-white fluorescent light providing cultures with 50,ttEcm-2s -t in a 12-12h photoperiod. Laminaria saccharina plants with mature sori were collected in Long Island Sound at Crane Neck, New York. A ripe portion of sorus was
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cut from the plant and wiped clean with 5% NaOCI (v/v in filtered seawater) and further rinsed with membrane filtered (Gelman) seawater. Meiospores were released, settled and germinated into gametophytes on coverslips in plastic Petri dishes. Mature gametophytes released gametes which underwent fertilization to produce zygotes. Copper treatmetat was applied on 3 days after inoculation when meiospores were settled and germinated. Specimens were examined under the light microscope and photographed. In another experiment, sporophytes sized above 1 cm long were placed in 5-liter plastic planting boxes, or l-liter beakers which contained five to ten plants, which were immersed in a refrigerated aquarium a t 12 + 1°C under cool-white fluorescent light with 20/~E c m - 2 s- ~ on a 1212 h photoperiod. The media were constantly agitated by wave plates in each planting box. Both boxes and beakers were aerated. The plants which had been cultured in the cold room were acclimated for 1 day and photographed before copper treatment. After 7 days of incubation in copper supplemented media, the plants were also photographed and hole punches using a No. 3 cork borer (154mm z) were taken for TEM and chlorophyll analysis. The blade area was measured with computerprogrammed graphic analysis from the photographs. Growth rates were monitored by the relative blade area increase. The specimens were cut into small pieces (less than l mm 2) and fixed, after incubation in 3% (v/v) glutaraldehyde plus 1% (w/v) paraformaldehyde (modified from Karnovsky, 1965), in 0'IM sodium cacodylate buffer (pH 7-2) or 0-05N phosphate buffer (pH 7-2) to which 3-5% (w/v) NaCI or 0-25M sucrose and 0.1% (w/v) caffeine (Muetler & Greenwood, 1978; Clayton & Beakes, 1983) with 0.05% (w/v) CaC1 z had been added, and post-fixed in 1% (w/v) OsO,, in the same buffer. After dehydration in EtOH or acetone, samples were embedded in Spurr's resin (Spurr, 1969). Thin sections were cut on a Porter Blum ultramicrotome, double stained with uranyl acetate and lead citrate and examined with a JEOL 100B TEM at 60kV. One disc was punched from the middle portion of each blade 5 cm above the blade-stipe junction using a No. 3 cork borer (154mm 2) and four discs were homogenized in 90% acetone with the addition of two drops of supersaturated MgCO 3 solution. After extraction, the samples were centrifuged and the supernatant analyzed using a spectrophotometer. The relative concentrations were calculated from the equations of Jeffrey & Humphrey (1975).
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RESULTS Abnormality in the very early stage of sporophytes Preliminary studies conducted on specimens which had been incubated in 50pg Cu per liter of media from mature gametophytes for 10 days showed several abnormal features in 4-20 celled sporophytes. A haptera-like protrusion appeared in the middle and/or at the distal end of the fronds (Fig. 1). The protrusions were different from true
1
Fig. 1. An abnormal growth pattern of a Laminaria saccharina sporophyte showing a haptera-likecell (arrow) at the end of a frond (bar = 20pm).
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2
Fig. 2. Someenlarged cells of Laminaria saccharina sporophytes in the middle (double arrow) and at the end of fronds (single arrow) (bar = 20 tim). haptera with respect to their location and morphology. Some cells were abnormally larger than others (Fig. 2) and some fronds showed dichotomous branching patterns in the middle or at the distal end of the fronds (Fig. 3). The 1-20 celled sporophytes which had been grown in the control media on the coverslip and then changed to media with copper showed growth inhibition in over 50 #g Cu per liter concentration after 10 days
3
Fig. 3. An abnormal growth pattern of a Laminaria saccharina sporophyte showing a dichotomous branching pattern in the middle of a frond (bar = 201Lm).
Boudewijn H. Brinkhuis, Ik Kyo Chung
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4
Fig. 4.
An abnormal growth pattern of a Laminaria saccharina sporophyte showing total loss of developmental polarity (bar = 20#m).
of incubation. The morphology of fronds was abnormal and similar to those of the preliminary study. However, the plants in lower copper concentrations continued their growth and development. Plants in the higher copper concentration remained in the filamentous shape and did not develop normally. Some showed irregular filamentous shapes (Fig. 4). These made large spherical, sometimes rounded pentagonal and hexagonal, cells in the fronds (Fig. 5) or had some bulges and protruberances (Fig. 6). These cells showed cell contents different from normal cells, which have peripheral plastids and a large central vacuole or big nucleus. There were many yellowish-green spheroid features which used to be chloroplasts in giant cells (Fig. 5). Some cells were lysed and lost cell contents. Uitrastructurai observation Normal structures
The nuclei of normal plant cells were enclosed in a limiting nuclear envelope consisting of two unit membranes separated by a perinuclear space. Normal ultrastructural features of LambTaria saccharina sporophytes showing the typical brown algal structure are shown in Figs 79. Chloroplast endoplasmic reticulum was not found. The Golgi apparatus was closely associated with the nuclear envelope (Fig. 7).
Copper effects on fine structure o1"L. saccharina
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Fig. 5. Some enlarged, rounded, hexagonal and pentagonal cells of Lamblaria saccharbut sporophytes showing small spheroid features in their cells which used to be chloroplasts (bar = 20/am).
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6
W Fig. 6. An abnormal growth pattern of Laminaria saccharina sporophytes showing enlarged cells, bulges and protuberances (arrows) (bar = 20#m).
Fig. 7. A nucleus, perinuclear Golgi dictyosomes and a chloroplast are shown together (bar = l # m ) . Note: ch = chloroplast, w = cell wall, G = Golgi dictyosome, m = mitochondria, n = nucleus, ph = physodes, v = vacuole.
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Fig. 8. A typical chloroplast and mitochondria are shown close to the cell wall (bar = l/~m). For explanation of abbreviations see caption to Fig. 7. Mitochondria had the same basic structure, with an outer smooth membrane surrounding an infolded inner membrane which enclosed a central space, the lumen or matrix. Some cristae were swollen at their tips (Fig. 8). Mitochondria showed various shapes (Fig. 9). They were scattered in the cytoplasm and occasionally some were adjacent to the chloroplasts. The Golgi apparatus consisted of individual stacks of cisternae, Golgi bodies or dictyosomes. The stack of parallel, disc-shaped cisternae forming the Golgi body were often curved. A network of tubules arises from the cisternal edges and these swelled in places to form different types of vesicles, so that the cisternae appeared fenestrated (Fig. 7). The chloroplast fine structure consisted of bands of three thylakoids, or discs, which traversed the whole length of each chloroplast and a peripheral band usually completely circled underneath the chloroplast envelope. An exchange of discs between adjacent bands often occurred in a regular pattern; a dichotomous branching pattern also was found. Membrane-free areas containing fibrous materials believed to be D N A material were tbund at each end of the chloroplast between the peripheral
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Fig. 9.
Boudewijn H. Brinkhui_~. lk Kvo Chung
A large osmophylic compound (physode) is shown in a vacuole (bar = 1l,rn). For explanation of abbreviations sec caption to Fig. 7.
band and the termination of the longitudinal bands (Fig. 8). Membranefree areas were also found between longitudinal bands in the middle of the chloroplast. Chloroplast envelopes were so tightly b o u n d to the peripheral thylakoid bands that they were hardly recognizable. No pyrenoids and no outpocketing of stroma was found. Affected features The most dramatic ultrastructural changes in young sporophytes treated in I00 and 5001~g Cu per liter of media for seven days were found in chloroplasts; these changes are shown in Figs 10--12 for Laminaria saccharina sporophytes treated in 500 !Lg Cu per liter of media for seven days. Slight swelling and detachment of thylakoid membranes seemed to be the first s y m p t o m of copper toxicity and the rupture and diffusion of the thylakoid membrane resulted in the regional swelling which caused the disruption of the parallel thylakoid band feature (Fig. 10).
Copper effects on fine structure of L. saccharina
Fig. tO.
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An affected chloroplast shows severe disintegration of the thylakoid band system plus undulating bands (bar = 1/~m).
The parallel pattern began to undulate in the affected area. Subsequently, totally degenerated chloroplast and remnants of thytakoid membrane were found in the chloroplast. Some chloroplasts showed remnants of thylakoid bands without chloroplast envelopes (Fig. l 1). Cytoplasmic features were also changed. Large central vacuoles were not found. There were several peculiar features which seemed to have central cores with peripheral network fibrils and they were connected by adjacent cores in the cytoplasm. These might be a diffused vacuole and/or membrane-bound vesicles because there were remnants of membraneous material. Although there were tubular networks around chloroplasts, it was not clear what they were. They might be deformed Golgi bodies or endoplasmic reticula (Fig. 12). There were some changes in the cell wall arrangement. Loose arrangement of fibrils in the inner cell wall region and somewhat larger
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Fig. 11.
Boudewijn H. Brinkhuis, Ik Kvo Chung
Aff~ted chloroplasts and totally diffused cytoplasmic contents found in an epidermal cell (bar = I gtm).
Fig. 12. Cytoplasmic features of an epidermal cell show some cores (arrows) and filamentous connections among cores and diffused organcllc contents. Tubular networks (arrowheads) around chloroplasts and large gap (*) in the cell wall are found (bar = t ~m).
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TABLE 1 Relative Chlorophyll Content in Untreated and Copper Treated Laminaria saccharina Sporophytes After 7 Days Treatment
Control 10/~g liter- t 50~g liter- ~ 100/~g liter -~ 500/ag liter- t
Per cent total chlorophyll
Per cent chlorophylls C 1+ C2
100 105.4 + 6.29 80.5 + 9.07 88-3+12.91 13.7 + 6'96
100 99-4 + 4-88 94.5 + 2-90 93.2+4-19 66.7 + 2.03
gaps were found in the cell wall (Fig. 12), which were different from the original intercellular space because of the remnant of fibrils in that area. Reduction in chlorophyll contents During the incubation period, small sporophytes ir~ 100 and 500~g Cu per liter media exhibited chlorosis and disintegration in the distal and marginal blade portions. Table 1 shows the relative total chlorophyll contents. The relative chlorophyll contents of 500/lg Cu per liter treated plants were significantly different from those in plants treated with other copper concentrations at the c~= 0"05 level. The relative concentrations of chlorophyll C t + C 2 in the control and lower copper concentration were not significantly different from each other at the ~. = 0-05 level (Table 1).
DISCUSSION Cellular responses to pollutant induced injury can provide rapid and highly sensitive indicators of environmental impacts. It may be possible to observe alterations in the structural-functional organization in individual target cells or groups of cells at an early stage of a reaction to cell injury. The abnormalities in the 4-20 celled sporophytes caused by copper treatment in this study are summarized in Fig. 13. The prominent features---enlargement of cells and protuberance--may be caused by disturbances in cell division processes, such as cytoplasmic division without cytokinesis, or loss of mitotic ability, or total disturbances in
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Fig. 13.
Boudewijn H. Brinkhuis, Ik Kyo Chung
e¢
Diagrammatic summary shows the abnormal growth patterns of Laminaria saccharina sporophytes.
metabolism. Several studies have shown such impacts of copper on cell division in phytoplankton (Steeman Nielsen & Wium-Anderson, 1970; Rosko & Rachlin, 1977; Anderson & Morel, 1978; Morel et al. 1979; Fisher et al., 1981). Kanda (1946) found abnormal growth patterns-haptera-like growth at the distal end-- in the early stage of Laminaria cichorioides sporophytes. Those are similar to the apical haptera-like transformation found in this study. Kanda did not mention the reason for that abnormality. Unfavorable conditions may cause such abnormal features during the early stage of sporophytes.
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A reduction in chlorophyll content is the characteristic symptom of copper toxicity (Table 1). In some cases, Cu-induced chlorosis is due to impeded uptake or translocation of Fe. It may be that Fe transport into plastids is affected but could also be due to the effects of Cu on the incorporation of Fe into the Fe-binding enzymes and electron transport proteins (Platt-Aloia et al., 1983). The recent work of Sandmann & B6ger (1980a, b) suggests an explanation for development of chlorosis in leaves of plants on cupriferous sites. It was found that Cu ÷ and Cu 2 ÷ ions block photosynthetic electron transport in Scenedesmus and in isolated chloroplasts of spinach. Measurements of absorption spectra of the cytochromes and fluorescence emission data were interpreted in terms of two sites of inhibition: on the oxdizing side of PSII and the reducing side of PSI. Copper ions may also be responsible for acceleration of peroxidative degradation of the lipids of the chloroplast membranes. It has been shown that Cu may catalyze the formation of hydroxyl radical and Fenton-type reactions that result in the destruction of unsaturated membrane fatty acids. If Cu accumulates to these levels, then chlorosis through breakdown of the thylakoids could occur rapidly. The features shown in Figs 10, 11 and 12 may .be the result of disturbances in lipid metabolism or breakdown of lipid in the thylakoid membrane. Tolerance to Cu has been recorded for a wide range of plant species (Woolhouse, 1983). Several mechanisms, such as modifications of the plasmamembrane to withstand copper-induced damage, exclusion or decrease of uptake of copper, immobilization of copper in cell walls, compartmentalization of copper in soluble and/or insoluble complexes and enzyme adaptations, have been recognized. Laminaria may be tolerant to copper in concentrations up to 10 or 50/~g liter- 1. Ultrastructure of plants incubated at, and below, 10/~g liter-~ did not show any change. Normal cells showed similar features described in investigations of other brown algae (Bouck, 1965; Bisalputra, 1966; Evans, 1966; 1974; Bourne & Cole 1968, Evans & Holligan, 1972a, b, Rawlence, 1973; Sideman & Schreiver, 1977; Pellegrini 1979, 1980). Those exposed to 100~tg Cu liter -1 or higher concentrations showed severe damage in cell walls and cytoplasmic contents, especially chloroplasts. Several authors have indicated that the toxic metals could be incorporated in cell wall, vacuole or physode. They may be able to withstand some copper concentration. However, the mechanisms for Cu detoxification have not been defined.
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The present information of the cytological effects of copper can have implications at the ecosystem level. The effect of copper on the early stages of L. saccharina may decrease the total primary production to failures in recruitment and decreased growth (Chung & Brinkhuis, in press). Abnormalities in the early life cycle stage, such as reduction in chlorophyll content and change in the fine structure, may have a potential deleterious impact on the primary production in the kelp ecosystem. These nearshore ecosystems are susceptible to coastal pollution sources of copper at levels within the range of values used in the present study (Chung & Brinkhuis, in press).
SUMMARY The present study demonstrates the following results: (1) Abnormal growth patterns of the early sporophytes--haptera-like protuberances, giant cells and abnormal branching patterns--were frequent in seawater copper concentrations > 50 ~g liter- t. (2) Ultrastructure of plants which exhibited chlorosis and abnormality showed changes in chloroplasts consisting of swollen thylakoids, denatured bands and a diffused matrix.
ACKNOWLEDGEMENTS This research was supported by the Gas Research Institute, New York State Energy Research and Development Authority, and the New York Gas Group by contracts to B.H.B. The research was conducted by I.K.C. as part of Master's thesis in Marine Environmental Studies at the State University of New York, Stony Brook.
REFERENCES Anderson, D. M. & Morel, F. M. M. (1978). Copper sensitivity of Goniaulax tamarensis. Limnol. Oceanogr., 23, 283-95. Babich, H. & Stotzky, G. (1985). Heavy metal toxicity to microbe-mediated ecologic processes: A review and potential application to regulatory policies. Environ. Res., 36, 111-37.
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Bisalputra, T. (1966). Electron microscope study of the protoplasmic continuity in certain brown algae. Can. J. Bot., 44, 89-93. Bouck, G. B. (1965). Fine structure and organelle association in brown algae. J. Cell Biol., 26, 523-37. Bourne, V. L. & Cole, K. (1968). Some observations on the fine structure of the marine brown alga Phaeostrophion irregulare. Can. J. Bot., 46, 136975. Brinkhuis, B. H., Breda, V. A., Tobin, S. & Macler, B. A. (1983). New York Marine Biomass Program---Culture of Laminaria saccharina. J. Worm Maricult. Soc., 14, 360-79. Brinkhuis, B. H., Mariani, E. C., Breda, V. A. & Brady-Campbell, M. M. (1984). Cultivation of Laminaria saccharina in New York Marine Biomass Program. HydrobioL, 116/117, 266-71. Bryan, G. W. (1983). Brown seaweed, Fucus vesiculosus, and the gastropod, Littorina littoralis, as indicators of trace-metal availability in estuaries. Sci. Tot. Environ., 28, 91-104. Bryan, G. W. & Hummerstone, L. G. (1973). Brown seaweed as an indicator of heavy metals in estuaries in south-west England. J. mar. biol. UK., 53, 705-20. Chung, I. K. & Brinkhuis, B. H. (in press). Copper effects in early stages of the kelp, Laminaria saccharina. Mar. Pollut. Bull. Clayton, M. M. & Beakes, G. W. (1983). Effects of fixatives on the ultrastructure ofphysodes in vegetative ceils ofScytosiphon lomentaria (Scytosiphonaceae, Phaeophyta). J. Phycol., 19, 4-16. Davies, A. G. (1976). An assessment of the basis of mercury tolerance in Dunaliella tertiolecta. J. mar. biol. Ass. UK., 56, 39-57. Evans, L. V. (1966). Distribution of pyrenoids among some brown algae. J. Cell. Sci., 1,449-54. Evans, L. V. (1974). Cytoplasmic organelles. In: Algalphysiology and biochemistry, (Stewart, W. D. P. (Ed.)), Cambridge University Press, 86-123. Evans, L. V. & Holligan, M. S. (1972a). Correlated light and electron microscopic studies on brown algae. I. Localization ofalginic and sulphated polysaccharides in Dictyota. New Phytol., 71, 1161-72. Evans, L. V. & Holligan, M. S. (1972b). Correlated light and electron microscopic studies on brown algae. II. Physode production in Dictyota. New Phytol., 71, 1173-80. Fisher, N. S., Jones, G. Y. & Nelson, D. M. (1981). Effects of copper and zinc on growth, morphology, and metabolism of Asterionella japonica (Cleve). J. exp. mar. Biol. Ecol., 51, 37-56. Haug, A., Melsom, S. & Omang, S. (1974). Estimation of heavy metal pollution in two Norwegian fjord areas by analysis of the brown alga Ascophyllum nodosum. Environ. Pollut., 7, 179-92. Jeffrey, S. W. & Humphrey, G. F. (1975). New spectrometric equations for determining chlorophylls a, b, cl and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanzen., 167, 191-4.
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Kanda, T. (1946). Culture studies of Laminariaceous plants in Hokkaido. J. Inst. Res. Sci. Fish. Hokodate Coll. Fish., 2, 1--44. (In Japanese.) Karnovsky, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol., 27, 137-8. Mann, K. H. (1972a). Ecological energetics of the seaweed zone in a marine bay on the Atlantic coast of Canada. I. Zonation and biomass of seaweed. Mar. Biol., 12, 1-10. Mann, K. H. (1972b). Ecological energetics of the seaweed zone in a marine bay on the Atlantic coast of Canada. II. Production of the seaweeds. Mar. Biol., 14, 199-209. Morel, F. E. M., Rueter, J. G., Anderson, D. M. & Guillard, R. R. L. (1979). Aquil: A chemically defined phytoplankton culture medium for trace metal studies. J. Phycol., 15, 135-41. Mortimer, D. C. & Czuba, M. (1982). Structural damage to leaf chloroplast of Elodea densa caused by methylmercury accumulated from water. Ecotoxicol. Environ. Safety, 6, 193-5. Mueller, W. C. & Greenwood, A. D. (1978). The ultrastructure of phenolicstoring cells fixed with caffeine. J. Exp. Bot., 29, 757-64. Myklestad, S., Eide, I. & Melsom, S. (1978). Exchange of heavy metals in Ascophyllum nodosum (L.) Le Jol. in situ by means of transplanting experiments. Environ. Pollut., 16, 277-84. Pellegrini, L. (1979). On the origin and development of vacuoles in promeristematic cells of Cystoseira stricta Sauvageau (Phaeophyta, Fucales). Protoplasrna, 101, 89-102. Pellegrini, L. (1980). Cytological studies on physoides in the vegetative cells of Cystoseira stricta Sauvageau (Phaeophyta, Fucales). J. Cell. Sci., 41,20931. Platt-Aloia, K. A., Thomson, W. W. & Terry, N. (1983).Changes on plastid ultrastructure during iron nutrition-mediated chloroplast development. Protoplasrna, 114, 85-92. Rawlence, D. J. (1973). Some aspects of the ultrastructure of Ascophyllum nodosum (L.) Le Jolls (Phaeophyceae, Fucales) including observations on cell plate formation. Phycologia, 12, 17-28. Rosko, J. J. & Rachlin, J. W. (1977). The effect of cadmium, copper, mercury, zinc and lead on cell division, growth and chlorophyll a content of the Chlorophyte Chlorella vulgaris. Bull. Torry Bot. Club., 104, 226-33. Sandmann, G. & B6ger, P. (1980a). Copper-mediated lipid peroxidation processes in photosynthetic membranes. Plant Physiol., 66, 797-800. Sandmann, G. & B6ger, P. (1980b). Copper deficiency and toxicity in Scenedesmus. Z. Pflanzenphysiol., 98, 53-9. Sicko-Goad, L. (1982). A morphometric analysis of algal response to low dose short-term heavy metal exposure. Protoplasma, 110, 75-86. Sicko-Goad, L. & Stoermer, E. F. (1979). A morphometric study of lead and copper effects on Diatoma tenue var. elongatum (Bascillariophyta). J. Phycol., 15, 316-21. Sideman, E. J. & Scheirer, D. C. (1977). Some fine structural observation on
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developing and mature sieve elements in the brown alga L. saccharina. Am. J. Bot., 64, 649-57. Silverberg, B. A. (1976). Cadmium-induced ultrastructural changes in mitochondria of freshwater green algae. Phycologia, 15, 155-9. Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastructure Research, 26, 31-43. Steeman Nielsen, E. & Wium-Anderson, S. (1970). Copper ions as poison in the sea and freshwater. Mar. Biol., 6, 93-7. Sunda, W. & Guillard, R. R. L. (1976). The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res., 34, 511-29. Thomas, W. H., Hollibaugh, J. J., Seibert, D. L. R. & Wallace, G. T., Jr. (1980). Toxicity of a mixture of ten metals to phytoplankton. Mar. Ecol. Prog. Ser., 2, 213-20. Woolhouse, H. W. (1983). Toxicity and tolerance in the response of plants to metals. In: Physiological plant ecology IlL (Lange, O. L., Nobel, P. S., Osmond, C. B. & Ziegler, H. (Eds)), Springer-Verlag. 245-300.
The Effects of Copper on the Fine Structure of the Kelp Laminaria saccharina (L.) Lamour* Boudewijn H. Brinkhuis & Ik K y o C h u n g Marine SciencesResearch Center, State Universityof New York, Stony Brook, New York 11794--5000, USA (Received: 28 February, 1986) A BSTRA CT The effects of copper on the fine structure of Laminaria saccharina were examined by incubating plants in 50, 100 and 500 #g Cu per liter of filtered and uv-treated seawater media. Copper treatment after fertilization in gametophytes yielded sporophytes that showed abnormal growth patterns, haptera-like protuburances, giant cells and abnormal branching patterns in 50 #g liter- t and 10012g liter-t copper additions. Ultrastructural examination of the plants which showed abnormality and chlorosis indicated the presence of denatured chloroplasts with swollen thylakoid membranes, detachment of thylakoid lamellae and diffused matrices.
INTRODUCTION Some temperate coastal ecosystems are based on seaweed primary productivity (Mann, 1972a, b). Bioaccumulated metals in seaweed tissue may be transferred to the detrital food web or directly to grazing organisms such as fish and sea urchins. The process of microbial activity in the sediment may be decreased by the metals accumulated in algal detritus. Finally, the total production of the system may be affected by the decreased microbial activity which recycles nutrients to the water column (Babich & Stotzky, 1985). Although there is still uncertainty about the processes involved in * Marine Sciences Research Center Contribution No. 503. 205 Marine Environ. Res. 0141-l136/86/$03-50 :~ ElsevierApplied SciencePublishers Ltd, England, 1986. Printed in Great Britain
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heavy metal accumulation by brown seaweeds, their ability to concentrate ions of metals from the surrounding seawater has been well documented and makes them useful indicators of marine heavy metal pollution (Bryan, 1983; Bryan & Hummerstone, 1973; Haug et al., 1974; Myklestad et al., 1978). The effects of accumulated metals on the plants themselves, however, have been less studied. One of the most commonly observed effects of heavy metal poisoning is a change in cell size or morphology. This has been observed in a wide variety of organisms. The uncoupling of cell growth and cell division (Davies. 1976) and interruption of cell separation, cytoplasmic changes in color and contents and disruption of chloroplast integrity and dispersion (Thomas et al., 1980), and morphological aberration (Sunda & Guillard, 1976) have been observed in phytoplankton species. Methylmercury absorbed by the aquatic plant, Elodea densa, affected chloroplast integrity (Mortimer & Czuba, 1982), which is very important to normal photosynthetic activity. Mortimer & Czuba proposed that the chloroplast membrane may be much more sensitive than other membranes within the cell. The ultrastructure of mitochondria also was affected by heavy metals. Cadmium treated freshwater green algae showed intra-mitochondrial dense granules and altered cristae structure (Silverberg, 1976). A significant decrease in the number of mitochondria in Diatoma species after gold treatment was based on the morphometric analysis (Sicko-Goad & Stoermer, 1979). Algal response to heavy metal exposure indicated that changes in vacuole volume were responsible for metal toxicity that was attributed to changes in membrane permeability (Sicko-Goad, 1982). Few such studies have been reported for seaweeds. The present study was conducted to delineate the morphological effects of copper on the brown alga, Laminaria saccharina, during early sporophyte stages.
MATERIALS AND METHODS The present study followed the culture methods of Brinkhuis et al. (1983, 1984). An incubation chamber was maintained at 10 + I°C and illuminated by 'very high output' cool-white fluorescent light providing cultures with 50,ttEcm-2s -t in a 12-12h photoperiod. Laminaria saccharina plants with mature sori were collected in Long Island Sound at Crane Neck, New York. A ripe portion of sorus was
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cut from the plant and wiped clean with 5% NaOCI (v/v in filtered seawater) and further rinsed with membrane filtered (Gelman) seawater. Meiospores were released, settled and germinated into gametophytes on coverslips in plastic Petri dishes. Mature gametophytes released gametes which underwent fertilization to produce zygotes. Copper treatmetat was applied on 3 days after inoculation when meiospores were settled and germinated. Specimens were examined under the light microscope and photographed. In another experiment, sporophytes sized above 1 cm long were placed in 5-liter plastic planting boxes, or l-liter beakers which contained five to ten plants, which were immersed in a refrigerated aquarium a t 12 + 1°C under cool-white fluorescent light with 20/~E c m - 2 s- ~ on a 1212 h photoperiod. The media were constantly agitated by wave plates in each planting box. Both boxes and beakers were aerated. The plants which had been cultured in the cold room were acclimated for 1 day and photographed before copper treatment. After 7 days of incubation in copper supplemented media, the plants were also photographed and hole punches using a No. 3 cork borer (154mm z) were taken for TEM and chlorophyll analysis. The blade area was measured with computerprogrammed graphic analysis from the photographs. Growth rates were monitored by the relative blade area increase. The specimens were cut into small pieces (less than l mm 2) and fixed, after incubation in 3% (v/v) glutaraldehyde plus 1% (w/v) paraformaldehyde (modified from Karnovsky, 1965), in 0'IM sodium cacodylate buffer (pH 7-2) or 0-05N phosphate buffer (pH 7-2) to which 3-5% (w/v) NaCI or 0-25M sucrose and 0.1% (w/v) caffeine (Muetler & Greenwood, 1978; Clayton & Beakes, 1983) with 0.05% (w/v) CaC1 z had been added, and post-fixed in 1% (w/v) OsO,, in the same buffer. After dehydration in EtOH or acetone, samples were embedded in Spurr's resin (Spurr, 1969). Thin sections were cut on a Porter Blum ultramicrotome, double stained with uranyl acetate and lead citrate and examined with a JEOL 100B TEM at 60kV. One disc was punched from the middle portion of each blade 5 cm above the blade-stipe junction using a No. 3 cork borer (154mm 2) and four discs were homogenized in 90% acetone with the addition of two drops of supersaturated MgCO 3 solution. After extraction, the samples were centrifuged and the supernatant analyzed using a spectrophotometer. The relative concentrations were calculated from the equations of Jeffrey & Humphrey (1975).
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RESULTS Abnormality in the very early stage of sporophytes Preliminary studies conducted on specimens which had been incubated in 50pg Cu per liter of media from mature gametophytes for 10 days showed several abnormal features in 4-20 celled sporophytes. A haptera-like protrusion appeared in the middle and/or at the distal end of the fronds (Fig. 1). The protrusions were different from true
1
Fig. 1. An abnormal growth pattern of a Laminaria saccharina sporophyte showing a haptera-likecell (arrow) at the end of a frond (bar = 20pm).
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2
Fig. 2. Someenlarged cells of Laminaria saccharina sporophytes in the middle (double arrow) and at the end of fronds (single arrow) (bar = 20 tim). haptera with respect to their location and morphology. Some cells were abnormally larger than others (Fig. 2) and some fronds showed dichotomous branching patterns in the middle or at the distal end of the fronds (Fig. 3). The 1-20 celled sporophytes which had been grown in the control media on the coverslip and then changed to media with copper showed growth inhibition in over 50 #g Cu per liter concentration after 10 days
3
Fig. 3. An abnormal growth pattern of a Laminaria saccharina sporophyte showing a dichotomous branching pattern in the middle of a frond (bar = 201Lm).
Boudewijn H. Brinkhuis, Ik Kyo Chung
210
4
Fig. 4.
An abnormal growth pattern of a Laminaria saccharina sporophyte showing total loss of developmental polarity (bar = 20#m).
of incubation. The morphology of fronds was abnormal and similar to those of the preliminary study. However, the plants in lower copper concentrations continued their growth and development. Plants in the higher copper concentration remained in the filamentous shape and did not develop normally. Some showed irregular filamentous shapes (Fig. 4). These made large spherical, sometimes rounded pentagonal and hexagonal, cells in the fronds (Fig. 5) or had some bulges and protruberances (Fig. 6). These cells showed cell contents different from normal cells, which have peripheral plastids and a large central vacuole or big nucleus. There were many yellowish-green spheroid features which used to be chloroplasts in giant cells (Fig. 5). Some cells were lysed and lost cell contents. Uitrastructurai observation Normal structures
The nuclei of normal plant cells were enclosed in a limiting nuclear envelope consisting of two unit membranes separated by a perinuclear space. Normal ultrastructural features of LambTaria saccharina sporophytes showing the typical brown algal structure are shown in Figs 79. Chloroplast endoplasmic reticulum was not found. The Golgi apparatus was closely associated with the nuclear envelope (Fig. 7).
Copper effects on fine structure o1"L. saccharina
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Fig. 5. Some enlarged, rounded, hexagonal and pentagonal cells of Lamblaria saccharbut sporophytes showing small spheroid features in their cells which used to be chloroplasts (bar = 20/am).
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6
W Fig. 6. An abnormal growth pattern of Laminaria saccharina sporophytes showing enlarged cells, bulges and protuberances (arrows) (bar = 20#m).
Fig. 7. A nucleus, perinuclear Golgi dictyosomes and a chloroplast are shown together (bar = l # m ) . Note: ch = chloroplast, w = cell wall, G = Golgi dictyosome, m = mitochondria, n = nucleus, ph = physodes, v = vacuole.
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Fig. 8. A typical chloroplast and mitochondria are shown close to the cell wall (bar = l/~m). For explanation of abbreviations see caption to Fig. 7. Mitochondria had the same basic structure, with an outer smooth membrane surrounding an infolded inner membrane which enclosed a central space, the lumen or matrix. Some cristae were swollen at their tips (Fig. 8). Mitochondria showed various shapes (Fig. 9). They were scattered in the cytoplasm and occasionally some were adjacent to the chloroplasts. The Golgi apparatus consisted of individual stacks of cisternae, Golgi bodies or dictyosomes. The stack of parallel, disc-shaped cisternae forming the Golgi body were often curved. A network of tubules arises from the cisternal edges and these swelled in places to form different types of vesicles, so that the cisternae appeared fenestrated (Fig. 7). The chloroplast fine structure consisted of bands of three thylakoids, or discs, which traversed the whole length of each chloroplast and a peripheral band usually completely circled underneath the chloroplast envelope. An exchange of discs between adjacent bands often occurred in a regular pattern; a dichotomous branching pattern also was found. Membrane-free areas containing fibrous materials believed to be D N A material were tbund at each end of the chloroplast between the peripheral
214
Fig. 9.
Boudewijn H. Brinkhui_~. lk Kvo Chung
A large osmophylic compound (physode) is shown in a vacuole (bar = 1l,rn). For explanation of abbreviations sec caption to Fig. 7.
band and the termination of the longitudinal bands (Fig. 8). Membranefree areas were also found between longitudinal bands in the middle of the chloroplast. Chloroplast envelopes were so tightly b o u n d to the peripheral thylakoid bands that they were hardly recognizable. No pyrenoids and no outpocketing of stroma was found. Affected features The most dramatic ultrastructural changes in young sporophytes treated in I00 and 5001~g Cu per liter of media for seven days were found in chloroplasts; these changes are shown in Figs 10--12 for Laminaria saccharina sporophytes treated in 500 !Lg Cu per liter of media for seven days. Slight swelling and detachment of thylakoid membranes seemed to be the first s y m p t o m of copper toxicity and the rupture and diffusion of the thylakoid membrane resulted in the regional swelling which caused the disruption of the parallel thylakoid band feature (Fig. 10).
Copper effects on fine structure of L. saccharina
Fig. tO.
215
An affected chloroplast shows severe disintegration of the thylakoid band system plus undulating bands (bar = 1/~m).
The parallel pattern began to undulate in the affected area. Subsequently, totally degenerated chloroplast and remnants of thytakoid membrane were found in the chloroplast. Some chloroplasts showed remnants of thylakoid bands without chloroplast envelopes (Fig. l 1). Cytoplasmic features were also changed. Large central vacuoles were not found. There were several peculiar features which seemed to have central cores with peripheral network fibrils and they were connected by adjacent cores in the cytoplasm. These might be a diffused vacuole and/or membrane-bound vesicles because there were remnants of membraneous material. Although there were tubular networks around chloroplasts, it was not clear what they were. They might be deformed Golgi bodies or endoplasmic reticula (Fig. 12). There were some changes in the cell wall arrangement. Loose arrangement of fibrils in the inner cell wall region and somewhat larger
216
Fig. 11.
Boudewijn H. Brinkhuis, Ik Kvo Chung
Aff~ted chloroplasts and totally diffused cytoplasmic contents found in an epidermal cell (bar = I gtm).
Fig. 12. Cytoplasmic features of an epidermal cell show some cores (arrows) and filamentous connections among cores and diffused organcllc contents. Tubular networks (arrowheads) around chloroplasts and large gap (*) in the cell wall are found (bar = t ~m).
Copper effects on fine structure of L. saccharina
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TABLE 1 Relative Chlorophyll Content in Untreated and Copper Treated Laminaria saccharina Sporophytes After 7 Days Treatment
Control 10/~g liter- t 50~g liter- ~ 100/~g liter -~ 500/ag liter- t
Per cent total chlorophyll
Per cent chlorophylls C 1+ C2
100 105.4 + 6.29 80.5 + 9.07 88-3+12.91 13.7 + 6'96
100 99-4 + 4-88 94.5 + 2-90 93.2+4-19 66.7 + 2.03
gaps were found in the cell wall (Fig. 12), which were different from the original intercellular space because of the remnant of fibrils in that area. Reduction in chlorophyll contents During the incubation period, small sporophytes ir~ 100 and 500~g Cu per liter media exhibited chlorosis and disintegration in the distal and marginal blade portions. Table 1 shows the relative total chlorophyll contents. The relative chlorophyll contents of 500/lg Cu per liter treated plants were significantly different from those in plants treated with other copper concentrations at the c~= 0"05 level. The relative concentrations of chlorophyll C t + C 2 in the control and lower copper concentration were not significantly different from each other at the ~. = 0-05 level (Table 1).
DISCUSSION Cellular responses to pollutant induced injury can provide rapid and highly sensitive indicators of environmental impacts. It may be possible to observe alterations in the structural-functional organization in individual target cells or groups of cells at an early stage of a reaction to cell injury. The abnormalities in the 4-20 celled sporophytes caused by copper treatment in this study are summarized in Fig. 13. The prominent features---enlargement of cells and protuberance--may be caused by disturbances in cell division processes, such as cytoplasmic division without cytokinesis, or loss of mitotic ability, or total disturbances in
218
Fig. 13.
Boudewijn H. Brinkhuis, Ik Kyo Chung
e¢
Diagrammatic summary shows the abnormal growth patterns of Laminaria saccharina sporophytes.
metabolism. Several studies have shown such impacts of copper on cell division in phytoplankton (Steeman Nielsen & Wium-Anderson, 1970; Rosko & Rachlin, 1977; Anderson & Morel, 1978; Morel et al. 1979; Fisher et al., 1981). Kanda (1946) found abnormal growth patterns-haptera-like growth at the distal end-- in the early stage of Laminaria cichorioides sporophytes. Those are similar to the apical haptera-like transformation found in this study. Kanda did not mention the reason for that abnormality. Unfavorable conditions may cause such abnormal features during the early stage of sporophytes.
Copper effects on fine structure of L. saccharina
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A reduction in chlorophyll content is the characteristic symptom of copper toxicity (Table 1). In some cases, Cu-induced chlorosis is due to impeded uptake or translocation of Fe. It may be that Fe transport into plastids is affected but could also be due to the effects of Cu on the incorporation of Fe into the Fe-binding enzymes and electron transport proteins (Platt-Aloia et al., 1983). The recent work of Sandmann & B6ger (1980a, b) suggests an explanation for development of chlorosis in leaves of plants on cupriferous sites. It was found that Cu ÷ and Cu 2 ÷ ions block photosynthetic electron transport in Scenedesmus and in isolated chloroplasts of spinach. Measurements of absorption spectra of the cytochromes and fluorescence emission data were interpreted in terms of two sites of inhibition: on the oxdizing side of PSII and the reducing side of PSI. Copper ions may also be responsible for acceleration of peroxidative degradation of the lipids of the chloroplast membranes. It has been shown that Cu may catalyze the formation of hydroxyl radical and Fenton-type reactions that result in the destruction of unsaturated membrane fatty acids. If Cu accumulates to these levels, then chlorosis through breakdown of the thylakoids could occur rapidly. The features shown in Figs 10, 11 and 12 may .be the result of disturbances in lipid metabolism or breakdown of lipid in the thylakoid membrane. Tolerance to Cu has been recorded for a wide range of plant species (Woolhouse, 1983). Several mechanisms, such as modifications of the plasmamembrane to withstand copper-induced damage, exclusion or decrease of uptake of copper, immobilization of copper in cell walls, compartmentalization of copper in soluble and/or insoluble complexes and enzyme adaptations, have been recognized. Laminaria may be tolerant to copper in concentrations up to 10 or 50/~g liter- 1. Ultrastructure of plants incubated at, and below, 10/~g liter-~ did not show any change. Normal cells showed similar features described in investigations of other brown algae (Bouck, 1965; Bisalputra, 1966; Evans, 1966; 1974; Bourne & Cole 1968, Evans & Holligan, 1972a, b, Rawlence, 1973; Sideman & Schreiver, 1977; Pellegrini 1979, 1980). Those exposed to 100~tg Cu liter -1 or higher concentrations showed severe damage in cell walls and cytoplasmic contents, especially chloroplasts. Several authors have indicated that the toxic metals could be incorporated in cell wall, vacuole or physode. They may be able to withstand some copper concentration. However, the mechanisms for Cu detoxification have not been defined.
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Boudewijn H. Brinkhuis, lk Kyo Chung
The present information of the cytological effects of copper can have implications at the ecosystem level. The effect of copper on the early stages of L. saccharina may decrease the total primary production to failures in recruitment and decreased growth (Chung & Brinkhuis, in press). Abnormalities in the early life cycle stage, such as reduction in chlorophyll content and change in the fine structure, may have a potential deleterious impact on the primary production in the kelp ecosystem. These nearshore ecosystems are susceptible to coastal pollution sources of copper at levels within the range of values used in the present study (Chung & Brinkhuis, in press).
SUMMARY The present study demonstrates the following results: (1) Abnormal growth patterns of the early sporophytes--haptera-like protuberances, giant cells and abnormal branching patterns--were frequent in seawater copper concentrations > 50 ~g liter- t. (2) Ultrastructure of plants which exhibited chlorosis and abnormality showed changes in chloroplasts consisting of swollen thylakoids, denatured bands and a diffused matrix.
ACKNOWLEDGEMENTS This research was supported by the Gas Research Institute, New York State Energy Research and Development Authority, and the New York Gas Group by contracts to B.H.B. The research was conducted by I.K.C. as part of Master's thesis in Marine Environmental Studies at the State University of New York, Stony Brook.
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