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Differential effects of Co2+ and Ni2+ on protein metabolism in Scenedesmus obliquus and Nitzschia perminuta
Environmental Toxicology and Pharmacology 16 (2004) 169–178
Differential effects of Co2+ and Ni2+ on protein metabolism in Scenedesmus obliquus and Nitzschia perminuta Mohamed E.H. Osman a , Amal H. El-Naggar a,∗ , Mostafa M. El-Sheekh a , Elham E. El-Mazally b a
Botany Department, Faculty of Science, Tanta University, Tanta, Egypt b Laboratories of Ministry of Health, Egypt Received 26 June 2003; accepted 17 December 2003
Abstract Growth, morphological changes, amino acid composition, total soluble protein, and protein electrophoretic pattern were monitored for Scenedesmus obliquus and Nitzschia perminuta grown in the presence of different concentrations of Co2+ and Ni2+ . Lower concentrations of cobalt stimulated the dry mass production and total soluble protein content of the two algae, whereas higher concentrations were inhibitory. Generally, N. perminuta showed more tolerance to the phytotoxicity of the two metals than S. obliquus and more tolerance to nickel than cobalt. However, S. obliquus seems to be more tolerant to cobalt than nickel. Cobalt and nickel have induced an increase in cell volume, change and disorder in cell shape. The increase in cell volume was much observed in Ni2+ treated cells. At the same time, the two metals did not induce any distinct morphological abnormalities in N. perminuta. Co2+ has stimulated the biosynthesis of all free amino acids in S. obliquus, except aspartic acid and phenylalanine, whereas Ni2+ caused 22% inhibition in the content of total free amino acids, except cystine and arginine. On the other hand, Co2+ has reduced the content of free amino acids in N. perminuta, except cystine, methionine, valine, and lysine. On the other hand, Ni2+ stimulated the biosynthesis of glycine, alanine and histidine and highly stimulated valine and sulphur containing amino acids (cystine and methionine) in N. perminuta. High cobalt concentration (4 ppm) resulted in the disappearance of 28.7 kDa protein, 3.5 ppm Ni2+ stimulated the appearance of 18 and 20 kDa proteins in S. Obliquus, while 37 kDa proteins disappeared from N. perminuta treated with high doses of Co2+ and Ni2+ . © 2004 Elsevier B.V. All rights reserved. Keywords: Cobalt; Nickel; Amino acids; Protein pattern; Scenedesmus; Nitzschia
1. Introduction Heavy metals are prevalent in municipal and industrial effluents, they modify the structure and productivity of aquatic ecosystems (Magdaleno et al., 1997). Heavy metals are known to disrupt algal metabolism either by inactivating the photosynthetic machinery, enzymatic pathways or by altering the nutrient transport and availability (Mallick and Rai, 1992; Rai et al., 1998). Heavy metals can be divided into two categories: essential and non essential (Reddy and Prasad, 1990). Cobalt is an essential micronutrients for all biota (Lustigman et al., 1995a). Within cells, it acts as component of Vitamin B12 and as essential cofactor in metalloenzymes (Munda and Hudnik, 1988). Manusadzianas
∗
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et al. (2002) considered cobalt as less toxic metal than Hg, Cu and Cd to the fresh water alga Nitellopsis obtusa. Lower concentrations of cobalt was found to increase dry weight, pigment fractions, total soluble proteins, nitrogen fixation, photosynthetic O2 -evolution and respiration of Calothrix fusca and Nostoc muscorum, whereas higher concentrations were inhibitory (El-Naggar et al., 1999). Moreover, high concentrations of cobalt were inhibitory for algal growth (Rachlin and Grosso, 1993), chlorophyll synthesis (Castorday et al., 1984; El-Naggar et al., 1999) and induced changes in photosynthetic activity (Tiwari and Mohanty, 1996). The toxic action of cobalt was found to be exerted at the level of plasma membrane by interacting with sulfhydryl groups on proteinaceous membranes to produce-s-metal-s-bridges which can alter membrane permeability (Rachlin and Grosso, 1993). Nickel is one of the toxic metals found in the various raw waste water of nonferrous metal, mineral processing,
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electronic electroplating, steel alloys (Lustigman et al., 1995b). It is highly mobile yet it is generally absorbed to only a small extent (Chan et al., 1991). Nickel caused reduction in the algal growth (Azazi and Mahmoud, 1988; Mallick et al., 1990;Wong and Wong, 1990); reduce NO3 − and NH4 + uptake (Rai and Raizada, 1989) and inhibit photosynthetic electron transport chain (Mallick and Rai, 1992; Rai et al., 1994a; El-Naggar, 1998). In a previous study, El-Naggar et al. (2002) have demonstrated that cobalt and nickel were present in relatively high concentrations in the water of river Nile at Kafr EL-Zayat city (an industrial city), which may affect structure and productivity of the phytoplankton communities of this location. Based on the above findings, the aim of the present study was to follow the direct effects of Co2+ and Ni2+ on the growth, cell morphology, amino acid composition, protein profile of Scenedesmus obliquus (unicellular green alga) and Nitzchia perminuta (unicellular diatom), which are dominating the river Nile at Kafr EL-Zayat city.
For the determination of protein electrophoretic pattern, the algal suspension containing 109 cells/ml was centrifuged at 3000 rpm for 5 min. The pellet was used for the extraction of protein by the method cited by Laemmli (1970) using an extraction buffer as recommended by Hawkesford and Belcher (1991) and described by El-Mazally (2002). The extract was used for protein estimation and electrophoretic pattern determination. The extract was diluted 20 times and the protein content was determined according to Lowry et al. (1951). The required calculations were conducted to obtain equal protein concentration in all samples (25 g/ml). The method of SDS vertical polyacrylamide gel electrophoresis (SDS–PAGE) was used as described by Laemmli (1970), for the determination of protein electrophoretic pattern. The gels were subjected to the staining solution for 1–2 h, followed by destaining solution over night. Thereafter, the destained gels were photographed while wet.
3. Results and discussion 2. Materials and Methods S. obliquus and N. perminuta were isolated from the Rosetta branch of river Nile at Kafer El-zayat City. The fresh water algal medium recommended by Kuhl (1962) was used for algal cultivation. Axenic cultures of the organisms were obtained by repeated subculturing and adding a mixture of streptomycin and tetracycline (30 ppm) to the medium for 20 min. In order to obtain sufficient algal cultures for the different investigations, the technique of mass culture (Lorenzen, 1964) was applied. The algal suspension was grown in 400 ml cylindrical pyrex glass vessels (50 cm in length and 4.5 cm in diameter) with narrow side tubes. The cultures were illuminated by means of fluorescent tubes (40 W.F. 7 day light), which gave light intensity of about 12 klux. The cultures were aereated with a mixture of 97% air and 3% CO2 . This mixture was allowed to pass through a series of bottles containing disinfecting solutions of CuSO4 /HgCl2 and finally through sterilized water. The algal growth was followed by the determination of the dry weight by the method used by Ahmed and Osman (1973). For the examination of the morphological changes, the algal samples were dehydrated with 5, 25, 50, 75, and 90% and absolute ethanol on a membrane filter (0.45 m pore diameter). The filters were air-dried, from which small pieces were mounted on SEM stubs, coated with gold then examined with a Jeol JSM-5300 scanning electron microscope (SEM). The total soluble proteins were determined by the method described by Lowry et al. (1951). Total free amino acids were determined according to Moore et al. (1958) using a Beckman amino acid analyzer (model 119 Cl). The quantity of each amino acid was calculated as gm amino acid/100 gm protein.
Low concentrations of cobalt (0.1 and 1 ppm) were found to increase the dry weight of S. obliquus throughout the experimental period reaching 9 and 16%, respectively, whereas high concentrations (2, 3 and 4 ppm) caused progressive reductions in the algal dry weight reaching 7, 33, and 39%, respectively at the end of the incubation period (Fig. 1). Similar results were more or less obtained with N. perminuta but the low concentrations required to induce growth stimulation were higher (0.5 and 1.5 ppm) than those required in S. obliquus (Fig. 2), where the higher concentrations, which caused growth inhibition ranged from 2.5–5 ppm. These results can indicate that N. perminuta being more resistant to Co2+ phytotoxicity than S. obliquus. These data are in agreement with those obtained by Lustigman et al. (1995a), who found that 10 and 20 ppm Co2+ caused a partial inhibition of growth in Chlamydomonas reinhardii, while 30 ppm and higher had prevented completely the algal growth. In addition, El-Naggar et al. (1999) found that lower Co2+ concentrations have stimulated the growth of N. muscorum, while higher ones were inhibitory. Other studies using Chlorella vulgaris indicated that the cells showed great resistance to Co2+ and that the toxic effects of the ions were exerted at the level of the plasma membrane (Rachlin and Grosso, 1993). Price and Morel (1990) have reported that growth promotion at low cobalt concentrations may due to cobalt substitution for Zn2+ in some metalloenzymes in vitro and in vivo. Low nickel concentrations (0.1 and 1 ppm) for S. obliquus and (0.5 and 2 ppm) for N. perminuta stimulated the dry weight production throughtout the experimental period. Higher doses of Ni2+ , (3.5 and 6.5 ppm) reduced the dry weight by 42 and 28% in S. obliquus and N. perminuta, respectively (Figs. 3 and 4). Similar results were observed in S. quadricauda by Angadi and Mathad (1994); in N. muscorum by Rai and Raizada (1986) and Rai and Raizada
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Fig. 1. Effect of different cobalt concentrations (ppm) on the dry weight of Scenedesmus obliquus.
(1989) and in Heamatococcus lacustris by Xylaender and Braune (1994). The inhibition of cell division by nickel exerted a strong influence on the dry weight gain (Gerhards and Weller, 1977). Generally, N. perminuta showed more tolerance to the two tested metals than S. obliquus and more tolerance to Ni2+ than Co2+ toxicity. However, S. obliquus was more tolerant to Co2+ than Ni2+ . The response to metal toxicity has been attributed to the differential affinity of various metal ions for sulfur complexation (Fisher and Jones, 1981). Again, the
metal tolerance was metal and genus dependent (El-Naggar, 1993). Since the morphological anomalous detected in the algal species can be used as bioindication of its habitat, it appears that cell volume and algal growth can be used interchangeably in toxicological tests. Therefore, studying the morphological changes induced by Co2+ and Ni2+ is one of great interest in our study. Results showed that Co2+ and Ni2+ have induced an increase in cell volume of S. obliquus cells compared with
Fig. 2. Effect of different cobalt concentrations (ppm) on the dry weight of Nitzschia perminuta.
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Fig. 3. Effect of different nickel concentrations (ppm) on the dry weight of Scenedesmus obliquus.
untreated cells (Fig. 5). However, the mean surface of S. obliquus exposed to Ni2+ was approximately twice that of control, whereas, Co2+ -treated cells were smaller than Ni2+ -treated cells but showing disorders in cell shape. Tukaj et al. (1998) demonstrated a good correlation between growth inhibition and the increase in mean cell volume in two Scenedesmus strains. Furthermore, Co2+ and Ni2+ induced change in shape and daughter cells liberation of S. obliquus cells. Daughter cells grow within mother cells but not released, leading to
an increase in mean cell volume. Similar findings were reported by Tukaj and Bohdanowicz (1995) for the morphology of three species of Scenedesmus as affected by aqueous fuel oil extract. Also, Yang (1993) reported the aberration of cell shape of six brown algae (the swelling and tendency to break) as a result of the inhibitory effects of germanium dioxide on growth. The latter author reported that germanium treatment can cause the malformation of fibrils in the cell wall. In the present study, higher concentrations of Co2+ and Ni2+ had no distinct morphological abnormalities on
Fig. 4. Effect of different nickel concentrations (ppm) on the dry weight of Nitzschia perminuta.
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Fig. 5. Scanning electron micrographs of Scenedesmus obliquus taken from early exponential phase (after 4 days of incubation). Plate 1: control of cells; plate 2: 4 ppm Co2+ -treated cells; and plate 3: 3.5 ppm Ni2+ -treated cells.
Fig. 6. Scanning electron micrographs of Nitzschia perminuta taken from early exponential phase (after 4 days of incubation). Plate 1: control cells; plate 2:5 ppm Co2+ -treated cells; and plate 3: 6.5 ppm Ni2+ -treated cells.
N. perminuta (Fig. 6), indicating that Nitzschia is resistant to morphological abnormalities, and this resistance may be due to the silicified nature of the cell wall. As indicated in Figs. 7 and 8, low Co2+ concentrations (0.1 and 1 ppm) for S. obliquus, and (0.5 and 1.5 ppm) for N. perminuta, induced significant increases in the protein content. Further increase in cobalt levels (2, 3 and 4 ppm for S. obliquus and 2.5, 3.5 and 5 ppm for N. perminuta) were associated with reductions in the protein content of the two tested algae. Similar trend was reported by El-Naggar et al. (1999) regarding the effect of cobalt and lead on the total soluble proteins of N. muscorum and C. fusca.
Nickel exerted a similar effect on the total soluble proteins of the two tested algae as that on the dry weight production (Figs. 9 and 10). The impact of Ni2+ on the total soluble proteins was observed earlier in some algal species (Stillwell and Holland, 1977; Angadi and Mathad, 1994; Xylaender and Braune, 1994). It could be suggested that accumulation of protein at low heavy metal concentrations may be one of the ways through which the algae can abolish their toxic effects, or to increase respiration leading to the utilization of carbohydrate in favor of protein accumulation. Such interpretation has been supported by the results obtained by El-Mazally (2002), who observed that low concentrations of Co2+ stimulated the respiration of these organisms. On the
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Fig. 7. Effect of different cobalt concentrations (ppm) on the total soluble protein of Scenedesmus obliquus.
other hand, inhibition of protein accumulation induced by higher concentrations of heavy metals may be attributed to the toxic action of these heavy metals on the enzymatic reactions responsible for protein biosynthesis (Hart and Scaife, 1997; Kobbia et al., 1985; Rai et al., 1994b; El-Naggar, 1993; Nassar, 2000). Co2+ (4 ppm) stimulated the biosynthesis of all amino acids in S. obliquus, except aspartic acid and phenylalanine (Table 1). The most pronounced stimulation (twoand seven-folds) was detected in proline and cystine, respectively. Such enhancement of proline content by metal
stress is a common metabolic response of algae (El-Naggar, 1993). On the other hand, Ni2+ induced inhibition (22%) in the total free amino acids of S. obliquus, except cystine and arginine which increased by 2.8-folds and 33%, respectively. The pronounced stimulation of cystine in response to Co2+ treatment, was found to be associated with stimulation in both glutamic acid and glycine contents. Such three amino acids are known as precursors of glutathione, one of the antioxidant enzymes detected in a higher rate in stressed algal cells (Randhawa et al., 2001). Thus, the increase in the contents of these amino acids may affect the
Fig. 8. Effect of different cobalt concentrations (ppm) on the total soluble protein of Nitzschia perminuta.
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Fig. 9. Effect of different nickel concentrations (ppm) on the total soluble protein of Scenedesmus obliquus.
rate of glutathione biosynthesis, having a role in protecting the algal cells from oxidative damage induced under metal stress (El-Shintinawy, 1999, 2001). Also, Poonguzhali and Rao (1998) reported the production of glutathione, cystine, glycine and glutamic acid by algae in response to metal stress, suggesting the possibility that phytochelatins were involved in metal binding.
On the other hand, the higher Co2+ concentration (5 ppm) in case of N. perminuta has resulted in the reduction of all amino acids content, except lysine. However, valine and cystine were highly stimulated (83% and nine-folds, respectively) by Co2+ , whereas methionine was slightly stimulated (Table 1). Leucine and valine are metabolically cross linked via their precursors pyruvate. It is well known that the
Fig. 10. Effect of different nickel concentrations (ppm) on the total soluble protein of Nitzschia perminuta.
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Fig. 11. The SDS–PAGE profile of total protein of Scenedesmus obliquus. Lane 1: control; lane 2: 0.1 ppm Co2+ -treated; lane 3: 4 ppm Co2+ -treated; lane 4: 0.1 ppm Ni2+ -treated; and lane 5: 3.5 ppm Ni2+ -treated cells.
alternative oxidase can be activated under stress, with the increase in the content of pyruvate. However, the first step in the biosynthetic pathway of valine and leucine involves acetohydroxy acid synthase. This enzyme has been inhibited under stress leading to alterations in the contents of valine, leucine and isoleucine (El-Shintinawy, 2001). Therefore, it is suggested that acetohydroxy acid synthase can be inactivated under Co2+ stress due to competition with alternative oxidase, resulting in the accumulation of valine at the expense of leucine in N. perminuta. At the same time, Ni2+ has stimulated the biosynthesis of glycine, alanine and histidine and highly stimulated valine and sulphur containing amino acids (cystine and methionine) in N. perminuta. The increase in sulphur containing amino acids in response to Ni2+ may reflect the strong affinity of Ni2+ to sulphur complexation as mentioned previously by El-Naggar (1993) with respect to Cu2+ and Cd2+ . In this regard, Maeda et al. (1990) detected ten-fold increase in cystine in the protein of Cd2+ -treated cells of C. vulgaris. In
Fig. 12. The SDS–PAGE profile of total protein of Nitzschia perminuta Lane1: control; lane 2: 0.5 ppm Co2+ -treated; lane 3: 5 ppm Co2+ -treated; lane 4: 0.5 ppm Ni2+ -treated; and lane 5: 6.5 ppm Ni2+ -treated.
addition, Ibrahim (1983) and Kobbia et al. (1985) observed an accumulation of cystine and arginine in Chlorella fusca in response to Ni2+ treatment. Similar observations were reported also in higher plants by Kastori et al. (1992), who found that Cu2+ and Cd2+ induced the accumulation of proline in Sunflower. Generally, the accumulation of amino acids in response to high Co2+ concentrations may lead to the assumption that suppressed protein biosynthesis encouraged free amino acids accumulation (Khalil, 1997) or may be due to some counteracting chelating mechanism against heavy metals toxicity (Woolhouse, 1983; Kobbia et al., 1985). The protein electrophoretic patterns of Scenedemus obliquus treated with low Co2+ concentrations did not show any significant changes compared with control. However, 4 ppm Co-treated S. obliquus showed the disappearance of 28.7 kDa band which was detected in the control track
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Table 1 Effect of cobalt and nickel on the amino acids composition of Scenedesmus obliquus and Nitzschia perminuta (calculated as g a.a./100 g dry weight) after 7 days of incubation Amino acids
Scenedesmus obliquus Content
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total
Cobalt (4 ppm)
Nitzschia perminuta Nickel (3.5 ppm)
Content
Cobalt (5 ppm)
Nickel (6.5 ppm)
6.99 2.95 2.72 6.01 3.28 2.92 5.14 0.08 3.91 1.20 4.97 5.44 2.07 3.12 1.86 4.24 3.85
6.88 3.65 2.80 6.86 6.75 3.10 6.04 0.62 4.18 1.29 6.54 5.44 2.08 2.69 1.87 4.74 4.20
5.06 2.60 2.00 5.10 3.07 2.46 3.54 0.23 2.82 0.86 3.12 3.26 1.32 1.89 1.66 3.28 5.12
3.57 2.11 1.95 3.57 3.07 1.91 2.25 0.06 0.18 0.65 2.43 2.19 0.97 1.43 0.88 1.81 2.11
3.46 2.01 1.88 3.17 2.87 1.82 2.11 0.11 1.61 0.67 1.91 1.76 0.77 1.40 0.75 2.33 1.71
2.67 1.58 1.09 2.85 1.82 1.97 2.36 1.96 1.89 1.28 1.94 2.14 0.80 1.39 1.34 1.74 1.57
60.75
69.73
47.39
31.14
30.34
30.39
(Fig. 11). In this context, Van Assche and Clijsters (1990) reported that Co2+ ions do not have a strong affinity for SH-groups, therefore secondary effects due to such interactions of the metal ions with proteins, if any, may be significantly minimized. However, S. obliquus cultures treated with 3.5 ppm Ni2+ exhibited two protein bands of 18 and 20 kDa, which were not originally detected in control track. The induction of low molecular weight proteins as a possible tolerance mechanism is a common response to metal stress in algae (Perez-Rama et al., 2001). On the other hand, protein electrophoretic pattern of Nitzschia perminuta showed that a band of about 37 kDa has disappeared in Co2+ - and Ni2+ -treated cultures (Fig. 12). In this connection, Poonguzhali and Rao (1998) detected the production of glutathione and amino acids like cystine, glycine and glutamine in response to metal stress and suggested the possibility that phytochelations were involved in metal binding. The latter authors showed that phytochelatins had molecular weights ranging from 10 to 30 kDa, with the predominant presence of cystine, glycine and glutamine. This finding may support the present results, where cystine, glycine and glutamic acid were accumulated in S. obliquus cells treated with 4 ppm Co2+ and the accumulation of both cystine and glycine in N. perminuta in response to nickel. References Ahmed, A.M., Osman, M.E.H., 1973. The influence of light on the CO2 fixation by synchronous cultures of Chlorella pyrenoidosa. Egypt. J. Bot. 16 (1–3), 319. Angadi, S.B., Mathad, P., 1994. Effect of chromium and nickel on Scenedesmus quadricauda (Turp.) de Breb. Phykos 33 (1/2), 99.
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Perez-Rama, M., Herrero Lopez, C., AbaldeAlonso, J., Torres Vaamonde, E., 2001. Class III metallothioneins in response to cadmium toxicity in the marine microalga Tetraselmis suecica (Kylin) Butch. Environ. Toxicol. Chem. 20 (9), 2061. Poonguzhali, T.V., Rao, V.N.R., 1998. Response of algae to metal pollution. Phykos 37 (1–2), 19. Price, N.M., Morel, F.M.M., 1990. Cadmium and cobalt substitution for zinc in marine diatoms. Nature 344, 656. Rachlin, J.W., Grosso, A., 1993. The growth response of the green alga Chlorella vulgaris to combined divalent cation exposure. Arch. Environ. Contam. Toxicol. 24 (1), 16. Rai, L.C., Raizada, M., 1986. Nickel induced stimulation of growth, heterocyst differentiation, C-14 uptake and nitrogenase activity in Nostoc muscorum. J. Indian Bot. Soc. 67, 74. Rai, L.C., Raizada, M., 1989. Effect of bimetallic combinations of Ni2+ , Cr3+ and Pb2+ on growth, uptake of nitrate and ammonia, CO2 fixation and nitrogenase activity of Nostoc muscorum. Ecotoxicol. Environ. Saf. 17, 175. Rai, L.C., Meena-Raizada, Singh, A.K., Kashyap, A.K., Kumar, H.D., 1994. Heavy metal toxicity in a nitrogen fixing cyanobacterium: interaction with sulfur containing amino acids and reducing substances, Rec. Adv. Phycol. 16, 265. Rai, P.K., Mallick, N.I., Rai, L.C., 1994b. Effect of nickel on certain physiological and biochemical behaviors of an acid tolerant Chlorella vulgaris. Biometals 7 (2), 193. Rai, L.C., Tyagi, B., Rai, P.K., Mallck, N., 1998. Interactive effect of UV-B and heavy metals (Cu and Pb) on nitrogen and phosphorus metabolism of N2-fixing canobacterium Anabaena doliolum. Environ. Exp. Bot. 39, 221. Randhawa, V.K., Zhou, F., Jin, X., Nalewaiko, C., Kushner, D.J., 2001. Role of oxidative stress and thiol antioxidant enzymes in nickel toxicity and resistance in strains of the green alga Scenedesmus acutus f. alternans. Can. J. Microbiol. 47 (11), 987. Reddy, G.N., Prasad, M.N.V., 1990. Heavy metal binding proteins peptides: occurrence, structure, synthesis and functions. Rev. Environ. Exp. Bot. 30, 251. Stillwell, E.F., Holland, J.R., 1977. Nickel effects on cell division, calcification and cell protein in the coccolithophorid Cricosphaera carterae. Sci. Biol. J. 3, 401. Tiwari, S., Mohanty, P., 1996. Cobalt induced changes in photosystem activity in Synechocystis PCC6803: alteration in energy distribution and stoichiometry. Photosynth. Res. 50, 243. Tukaj, Z., Bohdanowicz, J., Aksmann, A., 1998. A morphometric and stereological analysis of ultrastructural changes in two Scenedesmus (Chlorococcales, Chlorophyta) strains subjected to diesel fuel oil pollution. Phycologia 37 (5), 388. Tukaj, Z., Bohdanowicz, J., 1995. Diesel fuel oil induced morphological changes in some Scenedesmus species (Chlorococcales). Arch. Hydrobiol. Suppl. 108 (77), 83. Van Assche, F., Clijsters, H., 1990. Effects of metals on enzyme activity in plants. Plant Cell Environ. 13, 195. Woolhouse, H.W., 1983. Toxicity and Tolerance in the responses of plants to metals. In: Lange, O.L., Nobel, P.S., Osmound, B., Ziegler, H. (Eds.), Physiological Plant Ecology III. Encyclopaedia of Plant Physiology. New series, vol. 12C. Springer Verlag, Berlin, pp. 246– 289. Xylaender, M., Braune, W., 1994. Influence of nickel on the green alga Haematococcus lacustris rostafinski in phases of its life cycle. J. Plant Physiol. 144 (1), 86. Yang, W.X., 1993. Morphological study on the inhibitory effect of germanium dioxide on growth and development of brown algae. Sci. Pap. Inst. Algol. Res., Hokkaido. Univ. 9 (1), 33.
Differential effects of Co2+ and Ni2+ on protein metabolism in Scenedesmus obliquus and Nitzschia perminuta Mohamed E.H. Osman a , Amal H. El-Naggar a,∗ , Mostafa M. El-Sheekh a , Elham E. El-Mazally b a
Botany Department, Faculty of Science, Tanta University, Tanta, Egypt b Laboratories of Ministry of Health, Egypt Received 26 June 2003; accepted 17 December 2003
Abstract Growth, morphological changes, amino acid composition, total soluble protein, and protein electrophoretic pattern were monitored for Scenedesmus obliquus and Nitzschia perminuta grown in the presence of different concentrations of Co2+ and Ni2+ . Lower concentrations of cobalt stimulated the dry mass production and total soluble protein content of the two algae, whereas higher concentrations were inhibitory. Generally, N. perminuta showed more tolerance to the phytotoxicity of the two metals than S. obliquus and more tolerance to nickel than cobalt. However, S. obliquus seems to be more tolerant to cobalt than nickel. Cobalt and nickel have induced an increase in cell volume, change and disorder in cell shape. The increase in cell volume was much observed in Ni2+ treated cells. At the same time, the two metals did not induce any distinct morphological abnormalities in N. perminuta. Co2+ has stimulated the biosynthesis of all free amino acids in S. obliquus, except aspartic acid and phenylalanine, whereas Ni2+ caused 22% inhibition in the content of total free amino acids, except cystine and arginine. On the other hand, Co2+ has reduced the content of free amino acids in N. perminuta, except cystine, methionine, valine, and lysine. On the other hand, Ni2+ stimulated the biosynthesis of glycine, alanine and histidine and highly stimulated valine and sulphur containing amino acids (cystine and methionine) in N. perminuta. High cobalt concentration (4 ppm) resulted in the disappearance of 28.7 kDa protein, 3.5 ppm Ni2+ stimulated the appearance of 18 and 20 kDa proteins in S. Obliquus, while 37 kDa proteins disappeared from N. perminuta treated with high doses of Co2+ and Ni2+ . © 2004 Elsevier B.V. All rights reserved. Keywords: Cobalt; Nickel; Amino acids; Protein pattern; Scenedesmus; Nitzschia
1. Introduction Heavy metals are prevalent in municipal and industrial effluents, they modify the structure and productivity of aquatic ecosystems (Magdaleno et al., 1997). Heavy metals are known to disrupt algal metabolism either by inactivating the photosynthetic machinery, enzymatic pathways or by altering the nutrient transport and availability (Mallick and Rai, 1992; Rai et al., 1998). Heavy metals can be divided into two categories: essential and non essential (Reddy and Prasad, 1990). Cobalt is an essential micronutrients for all biota (Lustigman et al., 1995a). Within cells, it acts as component of Vitamin B12 and as essential cofactor in metalloenzymes (Munda and Hudnik, 1988). Manusadzianas
∗
Corresponding author. Tel.: +20-40-3317591; fax: +20-40-3350804. E-mail address: [email protected] (A.H. El-Naggar).
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et al. (2002) considered cobalt as less toxic metal than Hg, Cu and Cd to the fresh water alga Nitellopsis obtusa. Lower concentrations of cobalt was found to increase dry weight, pigment fractions, total soluble proteins, nitrogen fixation, photosynthetic O2 -evolution and respiration of Calothrix fusca and Nostoc muscorum, whereas higher concentrations were inhibitory (El-Naggar et al., 1999). Moreover, high concentrations of cobalt were inhibitory for algal growth (Rachlin and Grosso, 1993), chlorophyll synthesis (Castorday et al., 1984; El-Naggar et al., 1999) and induced changes in photosynthetic activity (Tiwari and Mohanty, 1996). The toxic action of cobalt was found to be exerted at the level of plasma membrane by interacting with sulfhydryl groups on proteinaceous membranes to produce-s-metal-s-bridges which can alter membrane permeability (Rachlin and Grosso, 1993). Nickel is one of the toxic metals found in the various raw waste water of nonferrous metal, mineral processing,
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electronic electroplating, steel alloys (Lustigman et al., 1995b). It is highly mobile yet it is generally absorbed to only a small extent (Chan et al., 1991). Nickel caused reduction in the algal growth (Azazi and Mahmoud, 1988; Mallick et al., 1990;Wong and Wong, 1990); reduce NO3 − and NH4 + uptake (Rai and Raizada, 1989) and inhibit photosynthetic electron transport chain (Mallick and Rai, 1992; Rai et al., 1994a; El-Naggar, 1998). In a previous study, El-Naggar et al. (2002) have demonstrated that cobalt and nickel were present in relatively high concentrations in the water of river Nile at Kafr EL-Zayat city (an industrial city), which may affect structure and productivity of the phytoplankton communities of this location. Based on the above findings, the aim of the present study was to follow the direct effects of Co2+ and Ni2+ on the growth, cell morphology, amino acid composition, protein profile of Scenedesmus obliquus (unicellular green alga) and Nitzchia perminuta (unicellular diatom), which are dominating the river Nile at Kafr EL-Zayat city.
For the determination of protein electrophoretic pattern, the algal suspension containing 109 cells/ml was centrifuged at 3000 rpm for 5 min. The pellet was used for the extraction of protein by the method cited by Laemmli (1970) using an extraction buffer as recommended by Hawkesford and Belcher (1991) and described by El-Mazally (2002). The extract was used for protein estimation and electrophoretic pattern determination. The extract was diluted 20 times and the protein content was determined according to Lowry et al. (1951). The required calculations were conducted to obtain equal protein concentration in all samples (25 g/ml). The method of SDS vertical polyacrylamide gel electrophoresis (SDS–PAGE) was used as described by Laemmli (1970), for the determination of protein electrophoretic pattern. The gels were subjected to the staining solution for 1–2 h, followed by destaining solution over night. Thereafter, the destained gels were photographed while wet.
3. Results and discussion 2. Materials and Methods S. obliquus and N. perminuta were isolated from the Rosetta branch of river Nile at Kafer El-zayat City. The fresh water algal medium recommended by Kuhl (1962) was used for algal cultivation. Axenic cultures of the organisms were obtained by repeated subculturing and adding a mixture of streptomycin and tetracycline (30 ppm) to the medium for 20 min. In order to obtain sufficient algal cultures for the different investigations, the technique of mass culture (Lorenzen, 1964) was applied. The algal suspension was grown in 400 ml cylindrical pyrex glass vessels (50 cm in length and 4.5 cm in diameter) with narrow side tubes. The cultures were illuminated by means of fluorescent tubes (40 W.F. 7 day light), which gave light intensity of about 12 klux. The cultures were aereated with a mixture of 97% air and 3% CO2 . This mixture was allowed to pass through a series of bottles containing disinfecting solutions of CuSO4 /HgCl2 and finally through sterilized water. The algal growth was followed by the determination of the dry weight by the method used by Ahmed and Osman (1973). For the examination of the morphological changes, the algal samples were dehydrated with 5, 25, 50, 75, and 90% and absolute ethanol on a membrane filter (0.45 m pore diameter). The filters were air-dried, from which small pieces were mounted on SEM stubs, coated with gold then examined with a Jeol JSM-5300 scanning electron microscope (SEM). The total soluble proteins were determined by the method described by Lowry et al. (1951). Total free amino acids were determined according to Moore et al. (1958) using a Beckman amino acid analyzer (model 119 Cl). The quantity of each amino acid was calculated as gm amino acid/100 gm protein.
Low concentrations of cobalt (0.1 and 1 ppm) were found to increase the dry weight of S. obliquus throughout the experimental period reaching 9 and 16%, respectively, whereas high concentrations (2, 3 and 4 ppm) caused progressive reductions in the algal dry weight reaching 7, 33, and 39%, respectively at the end of the incubation period (Fig. 1). Similar results were more or less obtained with N. perminuta but the low concentrations required to induce growth stimulation were higher (0.5 and 1.5 ppm) than those required in S. obliquus (Fig. 2), where the higher concentrations, which caused growth inhibition ranged from 2.5–5 ppm. These results can indicate that N. perminuta being more resistant to Co2+ phytotoxicity than S. obliquus. These data are in agreement with those obtained by Lustigman et al. (1995a), who found that 10 and 20 ppm Co2+ caused a partial inhibition of growth in Chlamydomonas reinhardii, while 30 ppm and higher had prevented completely the algal growth. In addition, El-Naggar et al. (1999) found that lower Co2+ concentrations have stimulated the growth of N. muscorum, while higher ones were inhibitory. Other studies using Chlorella vulgaris indicated that the cells showed great resistance to Co2+ and that the toxic effects of the ions were exerted at the level of the plasma membrane (Rachlin and Grosso, 1993). Price and Morel (1990) have reported that growth promotion at low cobalt concentrations may due to cobalt substitution for Zn2+ in some metalloenzymes in vitro and in vivo. Low nickel concentrations (0.1 and 1 ppm) for S. obliquus and (0.5 and 2 ppm) for N. perminuta stimulated the dry weight production throughtout the experimental period. Higher doses of Ni2+ , (3.5 and 6.5 ppm) reduced the dry weight by 42 and 28% in S. obliquus and N. perminuta, respectively (Figs. 3 and 4). Similar results were observed in S. quadricauda by Angadi and Mathad (1994); in N. muscorum by Rai and Raizada (1986) and Rai and Raizada
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Fig. 1. Effect of different cobalt concentrations (ppm) on the dry weight of Scenedesmus obliquus.
(1989) and in Heamatococcus lacustris by Xylaender and Braune (1994). The inhibition of cell division by nickel exerted a strong influence on the dry weight gain (Gerhards and Weller, 1977). Generally, N. perminuta showed more tolerance to the two tested metals than S. obliquus and more tolerance to Ni2+ than Co2+ toxicity. However, S. obliquus was more tolerant to Co2+ than Ni2+ . The response to metal toxicity has been attributed to the differential affinity of various metal ions for sulfur complexation (Fisher and Jones, 1981). Again, the
metal tolerance was metal and genus dependent (El-Naggar, 1993). Since the morphological anomalous detected in the algal species can be used as bioindication of its habitat, it appears that cell volume and algal growth can be used interchangeably in toxicological tests. Therefore, studying the morphological changes induced by Co2+ and Ni2+ is one of great interest in our study. Results showed that Co2+ and Ni2+ have induced an increase in cell volume of S. obliquus cells compared with
Fig. 2. Effect of different cobalt concentrations (ppm) on the dry weight of Nitzschia perminuta.
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Fig. 3. Effect of different nickel concentrations (ppm) on the dry weight of Scenedesmus obliquus.
untreated cells (Fig. 5). However, the mean surface of S. obliquus exposed to Ni2+ was approximately twice that of control, whereas, Co2+ -treated cells were smaller than Ni2+ -treated cells but showing disorders in cell shape. Tukaj et al. (1998) demonstrated a good correlation between growth inhibition and the increase in mean cell volume in two Scenedesmus strains. Furthermore, Co2+ and Ni2+ induced change in shape and daughter cells liberation of S. obliquus cells. Daughter cells grow within mother cells but not released, leading to
an increase in mean cell volume. Similar findings were reported by Tukaj and Bohdanowicz (1995) for the morphology of three species of Scenedesmus as affected by aqueous fuel oil extract. Also, Yang (1993) reported the aberration of cell shape of six brown algae (the swelling and tendency to break) as a result of the inhibitory effects of germanium dioxide on growth. The latter author reported that germanium treatment can cause the malformation of fibrils in the cell wall. In the present study, higher concentrations of Co2+ and Ni2+ had no distinct morphological abnormalities on
Fig. 4. Effect of different nickel concentrations (ppm) on the dry weight of Nitzschia perminuta.
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Fig. 5. Scanning electron micrographs of Scenedesmus obliquus taken from early exponential phase (after 4 days of incubation). Plate 1: control of cells; plate 2: 4 ppm Co2+ -treated cells; and plate 3: 3.5 ppm Ni2+ -treated cells.
Fig. 6. Scanning electron micrographs of Nitzschia perminuta taken from early exponential phase (after 4 days of incubation). Plate 1: control cells; plate 2:5 ppm Co2+ -treated cells; and plate 3: 6.5 ppm Ni2+ -treated cells.
N. perminuta (Fig. 6), indicating that Nitzschia is resistant to morphological abnormalities, and this resistance may be due to the silicified nature of the cell wall. As indicated in Figs. 7 and 8, low Co2+ concentrations (0.1 and 1 ppm) for S. obliquus, and (0.5 and 1.5 ppm) for N. perminuta, induced significant increases in the protein content. Further increase in cobalt levels (2, 3 and 4 ppm for S. obliquus and 2.5, 3.5 and 5 ppm for N. perminuta) were associated with reductions in the protein content of the two tested algae. Similar trend was reported by El-Naggar et al. (1999) regarding the effect of cobalt and lead on the total soluble proteins of N. muscorum and C. fusca.
Nickel exerted a similar effect on the total soluble proteins of the two tested algae as that on the dry weight production (Figs. 9 and 10). The impact of Ni2+ on the total soluble proteins was observed earlier in some algal species (Stillwell and Holland, 1977; Angadi and Mathad, 1994; Xylaender and Braune, 1994). It could be suggested that accumulation of protein at low heavy metal concentrations may be one of the ways through which the algae can abolish their toxic effects, or to increase respiration leading to the utilization of carbohydrate in favor of protein accumulation. Such interpretation has been supported by the results obtained by El-Mazally (2002), who observed that low concentrations of Co2+ stimulated the respiration of these organisms. On the
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Fig. 7. Effect of different cobalt concentrations (ppm) on the total soluble protein of Scenedesmus obliquus.
other hand, inhibition of protein accumulation induced by higher concentrations of heavy metals may be attributed to the toxic action of these heavy metals on the enzymatic reactions responsible for protein biosynthesis (Hart and Scaife, 1997; Kobbia et al., 1985; Rai et al., 1994b; El-Naggar, 1993; Nassar, 2000). Co2+ (4 ppm) stimulated the biosynthesis of all amino acids in S. obliquus, except aspartic acid and phenylalanine (Table 1). The most pronounced stimulation (twoand seven-folds) was detected in proline and cystine, respectively. Such enhancement of proline content by metal
stress is a common metabolic response of algae (El-Naggar, 1993). On the other hand, Ni2+ induced inhibition (22%) in the total free amino acids of S. obliquus, except cystine and arginine which increased by 2.8-folds and 33%, respectively. The pronounced stimulation of cystine in response to Co2+ treatment, was found to be associated with stimulation in both glutamic acid and glycine contents. Such three amino acids are known as precursors of glutathione, one of the antioxidant enzymes detected in a higher rate in stressed algal cells (Randhawa et al., 2001). Thus, the increase in the contents of these amino acids may affect the
Fig. 8. Effect of different cobalt concentrations (ppm) on the total soluble protein of Nitzschia perminuta.
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Fig. 9. Effect of different nickel concentrations (ppm) on the total soluble protein of Scenedesmus obliquus.
rate of glutathione biosynthesis, having a role in protecting the algal cells from oxidative damage induced under metal stress (El-Shintinawy, 1999, 2001). Also, Poonguzhali and Rao (1998) reported the production of glutathione, cystine, glycine and glutamic acid by algae in response to metal stress, suggesting the possibility that phytochelatins were involved in metal binding.
On the other hand, the higher Co2+ concentration (5 ppm) in case of N. perminuta has resulted in the reduction of all amino acids content, except lysine. However, valine and cystine were highly stimulated (83% and nine-folds, respectively) by Co2+ , whereas methionine was slightly stimulated (Table 1). Leucine and valine are metabolically cross linked via their precursors pyruvate. It is well known that the
Fig. 10. Effect of different nickel concentrations (ppm) on the total soluble protein of Nitzschia perminuta.
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Fig. 11. The SDS–PAGE profile of total protein of Scenedesmus obliquus. Lane 1: control; lane 2: 0.1 ppm Co2+ -treated; lane 3: 4 ppm Co2+ -treated; lane 4: 0.1 ppm Ni2+ -treated; and lane 5: 3.5 ppm Ni2+ -treated cells.
alternative oxidase can be activated under stress, with the increase in the content of pyruvate. However, the first step in the biosynthetic pathway of valine and leucine involves acetohydroxy acid synthase. This enzyme has been inhibited under stress leading to alterations in the contents of valine, leucine and isoleucine (El-Shintinawy, 2001). Therefore, it is suggested that acetohydroxy acid synthase can be inactivated under Co2+ stress due to competition with alternative oxidase, resulting in the accumulation of valine at the expense of leucine in N. perminuta. At the same time, Ni2+ has stimulated the biosynthesis of glycine, alanine and histidine and highly stimulated valine and sulphur containing amino acids (cystine and methionine) in N. perminuta. The increase in sulphur containing amino acids in response to Ni2+ may reflect the strong affinity of Ni2+ to sulphur complexation as mentioned previously by El-Naggar (1993) with respect to Cu2+ and Cd2+ . In this regard, Maeda et al. (1990) detected ten-fold increase in cystine in the protein of Cd2+ -treated cells of C. vulgaris. In
Fig. 12. The SDS–PAGE profile of total protein of Nitzschia perminuta Lane1: control; lane 2: 0.5 ppm Co2+ -treated; lane 3: 5 ppm Co2+ -treated; lane 4: 0.5 ppm Ni2+ -treated; and lane 5: 6.5 ppm Ni2+ -treated.
addition, Ibrahim (1983) and Kobbia et al. (1985) observed an accumulation of cystine and arginine in Chlorella fusca in response to Ni2+ treatment. Similar observations were reported also in higher plants by Kastori et al. (1992), who found that Cu2+ and Cd2+ induced the accumulation of proline in Sunflower. Generally, the accumulation of amino acids in response to high Co2+ concentrations may lead to the assumption that suppressed protein biosynthesis encouraged free amino acids accumulation (Khalil, 1997) or may be due to some counteracting chelating mechanism against heavy metals toxicity (Woolhouse, 1983; Kobbia et al., 1985). The protein electrophoretic patterns of Scenedemus obliquus treated with low Co2+ concentrations did not show any significant changes compared with control. However, 4 ppm Co-treated S. obliquus showed the disappearance of 28.7 kDa band which was detected in the control track
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Table 1 Effect of cobalt and nickel on the amino acids composition of Scenedesmus obliquus and Nitzschia perminuta (calculated as g a.a./100 g dry weight) after 7 days of incubation Amino acids
Scenedesmus obliquus Content
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total
Cobalt (4 ppm)
Nitzschia perminuta Nickel (3.5 ppm)
Content
Cobalt (5 ppm)
Nickel (6.5 ppm)
6.99 2.95 2.72 6.01 3.28 2.92 5.14 0.08 3.91 1.20 4.97 5.44 2.07 3.12 1.86 4.24 3.85
6.88 3.65 2.80 6.86 6.75 3.10 6.04 0.62 4.18 1.29 6.54 5.44 2.08 2.69 1.87 4.74 4.20
5.06 2.60 2.00 5.10 3.07 2.46 3.54 0.23 2.82 0.86 3.12 3.26 1.32 1.89 1.66 3.28 5.12
3.57 2.11 1.95 3.57 3.07 1.91 2.25 0.06 0.18 0.65 2.43 2.19 0.97 1.43 0.88 1.81 2.11
3.46 2.01 1.88 3.17 2.87 1.82 2.11 0.11 1.61 0.67 1.91 1.76 0.77 1.40 0.75 2.33 1.71
2.67 1.58 1.09 2.85 1.82 1.97 2.36 1.96 1.89 1.28 1.94 2.14 0.80 1.39 1.34 1.74 1.57
60.75
69.73
47.39
31.14
30.34
30.39
(Fig. 11). In this context, Van Assche and Clijsters (1990) reported that Co2+ ions do not have a strong affinity for SH-groups, therefore secondary effects due to such interactions of the metal ions with proteins, if any, may be significantly minimized. However, S. obliquus cultures treated with 3.5 ppm Ni2+ exhibited two protein bands of 18 and 20 kDa, which were not originally detected in control track. The induction of low molecular weight proteins as a possible tolerance mechanism is a common response to metal stress in algae (Perez-Rama et al., 2001). On the other hand, protein electrophoretic pattern of Nitzschia perminuta showed that a band of about 37 kDa has disappeared in Co2+ - and Ni2+ -treated cultures (Fig. 12). In this connection, Poonguzhali and Rao (1998) detected the production of glutathione and amino acids like cystine, glycine and glutamine in response to metal stress and suggested the possibility that phytochelations were involved in metal binding. The latter authors showed that phytochelatins had molecular weights ranging from 10 to 30 kDa, with the predominant presence of cystine, glycine and glutamine. This finding may support the present results, where cystine, glycine and glutamic acid were accumulated in S. obliquus cells treated with 4 ppm Co2+ and the accumulation of both cystine and glycine in N. perminuta in response to nickel. References Ahmed, A.M., Osman, M.E.H., 1973. The influence of light on the CO2 fixation by synchronous cultures of Chlorella pyrenoidosa. Egypt. J. Bot. 16 (1–3), 319. Angadi, S.B., Mathad, P., 1994. Effect of chromium and nickel on Scenedesmus quadricauda (Turp.) de Breb. Phykos 33 (1/2), 99.
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