Molecular Marine Biology and Biotechnology (1998) 7(2), 97–104
Cadmium sequestration in the marine macroalga Kappaphycus alvarezii
Graduate Program in Bi
ology, The Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong
Department of Biology, The Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong
Abstract We identified phytochelatins (PCs) as the major intracellular cadmium (Cd) chelators in the marine macroalga Kappaphycus alvarezii. Phytochelatin isolated from this alga showed a less complex thiol profile than that of yeast and higher plants. In the general structure of (Glu-Cys)n-Gly for PC, the n value was less than 3 for this alga. Algal PC-Cd complexes existed in the high molecular weight and low molecular weight forms. The high molecular weight form is rich in acid-labile sulfide. The PC content in algal fronds was directly correlated with the ambient Cd concentration as well as exposure time. After the Cd-treated algal fronds were transferred to Cd-free seawater, the algal PC content showed no significant change, while the total Cd content decreased.
Introduction Many marine organisms accumulate trace metals to significant concentrations (Eisler, 1981). The metal concentration detected in the tissues could be used to estimate the metal concentration in the ambient environment. Species with this function are bioindicators or biomonitors. Several studies have addressed the basic prerequisites of biomonitor species for trace metal contamination in aquatic envi-
ronments (Butler et al., 1971; Haug et al., 1974; Phillips, 1977; Phillips and Rainbow, 1989). The species should be sessile, thus being representative of the studied area. They should be hardy and capable of tolerating a high level of heavy metals. For easy identification and sampling, they should be abundant in the studied areas. Most importantly, there should be a simple correlation between the pollutant concentration detected in the tissues and the average ambient pollutant concentration. Several studies on trace metal accumulation by macroalgae suggest that many species could satisfy these basic prerequisites and serve as good biomonitors of trace metal pollution in the water column (Bryan, 1969, 1971, 1976; Bryan et al., 1985; Hu et al., 1996). Recently, intensive economic development along the coastal regions of southeast China has significantly increased the discharge of industrial wastes containing heavy metals into marine waters. Many coastal marine algae, including some commercially important species, are the first to be exposed to this type of pollution. Metals accumulated by macroalgae as well as the effect of heavy metals on the macroalgae population could have profound effects on the marine ecosystem. Cadmium, one of the most toxic and persistent trace metals, is found in high concentrations in wastes generated from zinc smelting, electroplating, and sewage treatment. In this study, we investigated the details of Cd uptake and sequestration in the marine macroalga Kappaphycus alvarezii, a widely cultivated species for carrageenan production in south China and the Philippines. Results Algal growth response and cadmium uptake We grew the algal fronds of K. alvarezii in seawater with added CdCl2 at different concentrations and monitored the algal growth rate. In the presence of CdCl2 concentrations up to 75 ?M, the algal fronds showed no effect from the added Cd. After treatment with 100 ?M CdCl2 for 14 days, we again
* Correspondence should be sent to this author at the above address, or by e-mail to firstname.lastname@example.org. 1998 Springer-Verlag New York Inc.
S. Hu and M. Wu
Figure 1. Total Cd accumulation by K. alvarezii. The algal fronds were treated with 100 ?M CdCl2. Data are means ± SD (n = 6).
observed no significant morphologic changes, but the algal growth as estimated by fresh weight gain was inhibited (data not shown). At Cd concentrations higher than 150 ?M, we observed rapid discoloration of the algal frond. We selected a 100-?M CdCl2 concentration for the study of intracellular Cd sequestration to minimize the effect of algal growth on Cd uptake. Figure 1 shows the amount of Cd accumulated by algal fronds treated with 100 ?M CdCl2. The Cd content detected in algal fronds was proportional to the exposure time. After growing in the Cdcontaining seawater for 9 days, the algal fronds accumulated Cd to a level of 285.4 ?g/g dry weight. Approximately 68% of the accumulated Cd was detected in the soluble extract. Identification of phytochelatin as the major intracellular cadmium chelator The soluble extract prepared from algal fronds treated with 100 ?M CdCl2 for 9 days was subjected to gel filtration chromatography on a Sephadex G50 column. Extract prepared from untreated algal fronds served as the control. Figure 2A shows typical separation profiles. The Cd-treated sample contained two more peaks, which contained more than 80% of the Cd detected in the soluble extract (Figure 2B). These peaks were designated as peaks A and B. When the soluble extract from untreated al-
Figure 2. Gel filtration chromatography of soluble cell extract on a column of Sephadex G-50. (A) The A254nm elution profile of soluble extracts prepared from control algal fronds (dotted line) and from algal fronds treated with 100 ?M CdCl2 for 9 days (solid line). Peak positions of molecular weight standards are marked (arrows). (B) The Cd content and the A254nm profile of the soluble extract prepared from Cd-treated algal fronds.
gal fronds was mixed with an equivalent amount of CdCl2, we detected Cd in a peak centered at fraction 62 (data not shown). Therefore, peaks A and B represent Cd-binding compounds induced by Cd treatment. Compared with markers of known molecular weight, we estimated the apparent molecular weights of peaks A and B to be 5.1 and 3.5 kDa, respectively. Fractions at the leading edge of peak A, fractions 41–43, and the trailing edge of peak B, fractions 52–54, were acidified and subjected to highperformance liquid chromatography (HPLC) analysis as described under Experimental Procedures. Figure 3 shows the typical profile after postcolumn derivatization with DTNB (see Experimental Procedures) to detect sulfhydryl-containing compounds. We detected two major peaks and two minor peaks. We purified the major peaks for amino acid com-
Cadmium sequestration in macroalga
Table 1. Amino acid compositions of purified peaks 1 and 3 in Figure 3. Peak 1 (%) Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val 0.3 0 0 0.5 36.7 0 38.3 22.0 0.2 0.2 0.3 0.2 0 0.2 0.2 0.6 0.2 0 0 0.2 Peak 3 (%) 1.1 0.7 0 1.5 36.3 0 37.7 16.0 0.3 0.6 1.2 0.7 0.2 0.6 0 1.5 0.8 0 0 0.8
Figure 3. HPLC chromatograms show the thiol profile after the derivatization with DTNB. The thin line shows a fraction from peak A, and the thick line shows a fraction from peak B in Figure 2.
position analyses. The result showed that in both peaks, Glu, Cys, and Gly were the predominant components; other amino acids existed only at tract levels (Table 1). The ratio of Glu:Cys:Gly is 1.74: 1.67:1 for peak 1 and 2.36:2.27:1 for peak 3. On the basis of the isolation procedure, the amino acid composition, and the tight association with Cd, we identified the major Cd ligands in this macroalga as PCs. Phytochelatins possess the general structure of ( -Glu-Cys)n-Gly. They have been identified as the primary metal chelators in several species of higher plants, microalgae, and fungi (Murasugi et al., 1981; Grill et al., 1985, 1987; Gekeler et al., 1988). In these organisms, the reported n value ranged from 2 to 11. Characterization of the PC-Cd complexes The elution pattern (Figure 2) of algal PC-Cd complex on the Sephadex G-50 column showed some similarity to that of yeast and some higher plants (Murasugi et al., 1983; Reese and Winge, 1988; Speiser and Abrahamson, 1992). Upon acidification, peak A released the characteristic odor of H2S and indicated the possible presence of acid-labile sulfide. Therefore, we determined the acid-labile sulfide content in each fraction. Figure 4 shows that fractions in peak A contain much higher levels of acid-labile sulfide than fractions in peak B. Peaks A and B are thus analogous to the higher molecular weight and lower molecular weight complexes reported for Schizosaccharomyces pombe (Murasugi et al., 1983; Reese and
Winge, 1988). The S?2/Cd ratio of fractions 41–43 and 52–54 was determined to be 0.21 and 0.03, respectively (Table 2). The S?2/Cd ratio of high mo-
Figure 4. Acid-labile sulfide content and A254nm profile of the Cd-binding complexes purified by Sephadex G-50 chromatography. Peaks A and B correspond to the high and low molecular weight complexes described in the text.
S. Hu and M. Wu
Table 2. The S?2/Cd ratios in selected fractions of peaks A and B purified by gel filtration chromatography. High molecular weight Fraction 41 42 43 S?2/Cd 0.21 ± 0.01 0.22 ± 0.01 0.20 ± 0.01 Low molecular weight Fraction 54 55 56 S?2/Cd 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00
lecular weight fractions was much higher than that of low molecular weight fractions. The S?2/Cd ratio of 0.21 in the algal high molecular weight fraction was lower than that of tomato cell and S. pombe, with ratios of 0.35 and 0.6, respectively. This might indicate the structural difference among high molecular weight complexes isolated from different species. This assumption was partially substantiated by the different UV transition profiles observed in these complexes. The size of the CdS crystallite in the Cd-PC complex dictates the optical property of the complex. The smaller clusters should show more blue-shift in their electronic transition. The complexes purified from Candida glabrata and S. pombe contained 20-? crystallites and exhibited transitions between 300 and 315 nm (Dameron et al., 1989). The high molecular weight complex from K. alvarezii showed UV transition around 260 nm (Figure 5). The low molecular weight complex with a low content of acid-labile sulfide did not exhibit such transition. Upon acidification to pH 1.5, the UV absorbance diminished in both high and low molecular weight complexes and indicated the possible metal ion displacement induced by proton. The spectrum detected a reneutralized sample of the acidified low molecular weight complex was similar to that before acidification. The reneutralized high molecular weight complex did not recover the transition at 260 nm; rather, it exhibited the spectrum characteristic of the low molecular weight complex. The loss of 260 nm transition as a result of acidification could be directly correlated with the loss of acid-labile sulfide and the change of CdS cluster structure. Changes in cellular phytochelatin content in response to ambient cadmium concentration We estimated the total cellular PC content by measuring the total concentration of -Glu-Cys subunit.
Figure 5. Ultraviolet absorption spectra of the PC-Cd complexes purified by two rounds of Sephadex G-50 gel filtration chromatography: (A) shows the absorption spectra of high and low molecular weight fractions before acidification; (B) shows the absorption spectra after acidification to pH 2.0 and reneutralization to pH 7.0 for high molecular weight (solid line) and low molecular weight (dotted line) fractions.
Our detection method could not detect -Glu-Cys subunit in algal fronds grown in seawater without added Cd. After treatment with 50 ?M Cd for 9 days, we detected PC content at 1.31 ?mol/g dry weight. Treatment with 100 ?M Cd significantly enhanced PC accumulation to 4.28 ?mol/g dry weight (Figure 6A). Therefore, PC accumulation in K. alvarezii was directly related to the ambient metal concentration. In algal fronds treated with 100 ?M Cd for 9 days, we observed a linear increase of PC content over the exposure time (Figure 6B). The cellular level of PC in K. alvarezii was therefore a function of exposure time. We also examined the stability of PC in K. alvarezii. After treatment with 100 ?M Cd for 9 days, we washed the algal fronds, transferred them to Cd-free seawater, and continuously monitored PC content. We did not detect any significant change of the cellular PC level after 18 days (Figure 7A), while the algal fronds lost 11% of the total Cd ac-
Cadmium sequestration in macroalga
Figure 6. Phytochelatin accumulation in algal fronds treated with CdCl2: (A) algal fronds were treated with 50 ?M or 100 ?M CdCl2 for 9 days; (B) algal fronds were treated with 100 ?M CdCl2 for 3, 6, and 9 days. Data are means ± SD (n = 6).
cumulated (Figure 7B). We assumed this loss was due mainly to the Cd released from algal surface.
Discussion The red alga K. alvarezii showed Cd tolerance in the range of other commonly encountered seaweeds along the coastal areas of China (Hu et al., 1996). Algal growth was not affected by the presence of 50 ?M Cd, which inhibited the growth of many higher plants. The algal fronds could accu-
Figure 7. Change of PC content in Cd-treated algal fronds. The algal fronds were treated with 100 ?M CdCl2 days, then transferred to Cd-free seawater and grown for 18 days. Data are means ± SD (n = 6). Inset shows the change in total Cd content.
mulate Cd up to 285 ?g/g dry weight. The binding of Cd to algal cell wall definitely accounted for a portion of the total accumulation. Metal adsorption to the cell wall material was reported as a process approximating ionic exchange. Many studies have noted the high affinity of trace metals for polysaccharides such as alginates and carrageenan, which are essential components in the cell walls of brown and red algae, respectively (Haug, 1961; Veroy et al., 1980; Kuyucak and Voleskey, 1989a, 1989b). In several species of brown algae, most accumulated metals have been found in the intracellular components and associated principally with polyphenols (Skipnes et al., 1975; Ragan et al., 1979; Bryan and Gibbs, 1983; Karez and Pereira, 1995). In this study, we identified PC as the major intracellular Cd chelator in the red alga K. alvarezii. We also demonstrated that algal PC existed in both high and low molecular weight forms. Algal PC had the same general structure of (Glu-Cys)n-Gly. Phytochelatin isolated from this alga possessed a less complex thiol profile and lower n value than that of yeast and higher plants. Phytochelatin was first discovered in the fission yeast (Murasugi et al., 1981). Functionally analogous to metallothioneins in animal cells, PC can detoxify intracellular metal by chelating the metal ions through coordination with sulfhydryl groups. A variety of metals, including Cd, Cu, Zn, Pb, Hg, Ni, Bi, Ag, and Au, induce PC synthesis. Two types of Cd-PC complexes, referred as high molecular weight and low molecular weight, were first reported in S. pombe (Murasugi et al., 1983). The acid-labile sulfide content in the high molecular weight complex was significantly higher than that in the low molecular weight complex. The importance of acid-labile sulfide and high molecular weight complex in metal detoxification was substantiated by genetic studies. In S. pombe, the Cdhypersensitive mutants were deficient in high molecular weight production (Mutoh and Hayashi, 1988; Speiser et al., 1992). The high and low molecular weight complexes were also isolated from higher plants, Brassica juncea and Lycopersicon esculentum (Reese et al., 1992; Speiser and Abrahamson, 1992). Our study is the first to demonstrate the presence of high and low molecular weight PC-Cd complexes in algal species. The effective usage of acid-labile sulfide for Cd sequestration as indicated by the low S?2/Cd ratio detected in the algal high molecular weight fraction might contribute to the significant Cd tolerance observed in this alga.
S. Hu and M. Wu
Heavy metals are the primary inducers of PC synthesis. Although Cd has induced the synthesis of heat shock proteins (Schollf and Key, 1982; Czarnecka et al., 1984), heat shock treatment did not induce PC synthesis. Although CuSO4 effectively elicited the synthesis of both phytoalexins and PC, elicitor treatments, such as UV radiation or contact with fungal cell wall, did not induce PC synthesis. Cold shock, alteration of hormonal levels, and oxidative stresses did not induce PC synthesis in cell cultures (Steffens, 1990). Therefore, the tight regulation of PC synthesis by metal ions makes PC content a suitable parameter for monitoring heavy metal impact. After exposure to a sublethal level of Cd, the intracellular PC content in K. alvarezii showed a dose-dependent induction kinetics. The intracellular PC content showed a good correlation with the length of exposure to Cd. Several lines of evidence have demonstrated that in tobacco cells the PC-Cd complexes are compartmentalized into vacuoles (Vogeli-Lange and ¨ Wagner, 1990). But the fate and turnover of the intracellular PC were not studied in this system. In the Cd-exposed marine diatom Thalassiosira weissflogii, the cells exported a small amount of PC when they were suspended in Cd-free medium. The same author detected continuous PC export when the cells were kept in Cd-containing medium (Lee, 1996). The degradation of PCs was observed in Silene vulgaris after the Cd exposure was terminated (Knecht et al., 1995). In this study, we detected that in K. alvarezii the PC level remained at a relatively constant level, while the total Cd accumulation decreased after the Cd stress was removed for 18 days. The tolerance to a high concentration of Cd and stable maintenance of PC in K. alvarezii suggests that the alga has the potential to be used as a scavenger to collect and accumulate Cd present in polluted marine environments.
ter. Complete cell breakage was achieved by grinding with a mortar and pestle while frozen with liquid nitrogen. The fine powders were quickly transferred to 5 ml of 10 mM Tris-HCl buffer (pH 8.5). The mixture was homogenized by ultrasonication (B. Braun Labsonic U) for 5 min with a repeating duty cycle of 0.5 s. The homogenates were centrifuged at 12,000 g for 15 min at 4°C to remove coarse debris. The supernatant was collected and subjected to a second round of centrifugation at 100,000 g for 30 min at 4°C. The clear supernatant was collected for subsequent analysis. Isolation, purification, and characterization of cadmium-binding components Two milliliters of soluble extract containing approximate 2 mg of protein was loaded on a Sephadex G-50 column (2.5 × 70 cm) equilibrated with 0.1 M KCl, 20 mM Tris-HCl, pH 7.8. The column was developed at 4°C with the same buffer at a flow rate of 1 ml/min, the 254-nm absorbance profile was recorded. The eluate was collected in 5-ml fractions. A portion (200 ?l) of each fraction was used for Cd measurement by atomic absorption spectrophotometry (Z-8100, Hitachi). The Cdcontaining fractions of each peak were pooled, lyophilized, and used for HPLC (Waters) analysis. The freeze-dried residue was dissolved in 0.2% trifluoroacetic acid (TFA) and injected to a 4.6 × 250-mm reverse-phase column (LKB TSK ODS120T, 5 ?m). Separation was carried out by using the solvent system of 0% to 20% acetonitrile gradient in 0.1% TFA. The developing time was 40 min. The column effluent was derivatized with 5, 5 -dithiobis(-2-nitrobenzoic acid) (DTNB), and the SH-containing fractions were specifically detected at 412 nm (Grill et al., 1985). For further purification, individual peaks were collected and subjected to a repeated round of separation. Each peak fraction was again collected, desalted, and lyophilized. The amino acid composition for each peak was performed at Mann Laboratory, University of California at Davis. Cysteine content was estimated by performic acid oxidation and cysteic acid determination. Quantitative analysis of the phytochelatins The acidified supernatant obtained by using 10% sulfosalicylic acid (SSA), instead of the Tris-HCl buffer, in the extraction process was used for quantitative analysis. The extraction procedure was the same as described above. The acidified extracts
Experimental Procedures Algal cultivation, determination of cadmium content We obtained K. alvarezii from a coastal aquaculture farm in Hainan, China. The algal culture conditions and the Cd analysis procedure were described in our previous study (Hu et al., 1996). Preparation of the soluble cell extracts Approximately 20-g (fresh weight) algal fronds were cleaned and lyophilized to remove excess wa-
Cadmium sequestration in macroalga
were subjected to the same HPLC separation procedure. After postcolumn derivatization with DTNB, the specific absorbance at 412 nm for SHcontaining compounds was quantified by comparison with peak areas of glutathione (GSH) standards (Sigma Chemical Co.). By this method we were able to detect the presence of 1 nmol PC. Determination of the acid-labile sulfide Acid-labile sulfide was measured according to Rabinowitz (1978). We added 0.5 ml of 1 M ZnOAc and 0.1 ml of 6% (wt/vol) NaOH sequentially to 0.5 ml of eluate from each fraction. After vortexing, we added 0.25 ml of N, N -dimethyl-p-phenylene diamine HCl solution (0.1% in 6 N HCl) and swirled the samples until clear. After adding 0.1 ml of FeCl3 stock solution (0.31% in 0.6 N HCl), the sample was mixed by vortexing. After incubation at room temperature for 30 min, we measured the absorbance at 670 nm in a Beckman DU-650 spectrophotometer. Na2S served as the calibration standard. Ultraviolet spectroscopy Materials collected from the leading edge of high molecular weight and the trailing edge of low molecular weight peaks were purified by two rounds of gel filtration on the Sephadex G-50 column. Spectroscopic analysis of these materials was conducted using a Beckman DU-650 spectrophotometer. The Cd-binding complexes were acidifed and reneutralized by titration with 0.6 N HCl and 0.6 N NaOH, respectively.
Acknowledgments This research has been supported by a grant from the Biotechnology Research Institute, The Hong Kong University of Science and Technology.
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