- Email: [email protected]
Speciation of cadmium–γ-glutamyl peptides complexes in cells of the marine microalga Phaeodactylum tricornutum
Plant Science 163 (2002) 807 /813 www.elsevier.com/locate/plantsci
Speciation of cadmium g-glutamyl peptides complexes in cells of the marine microalga Phaeodactylum tricornutum /
Elisabetta Morelli a, Boris H. Cruz b, Simone Somovigo a, Gioacchino Scarano a,* a
Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Area della Ricerca di Pisa, Via G. Moruzzi 1, 56124 Pisa, Italy b Departament de Quimica Analitica, Universitat de Barcelona, Av. Diagonal 647, E 08028 Barcelona, Spain Received 17 May 2002; received in revised form 2 July 2002; accepted 3 July 2002
Abstract Phaeodactylum tricornutum responds to Cd exposure by synthesizing phytochelatins (PCn ) [(g-Glu /Cys)n /Gly] with n values from 2 to 6. Size exclusion chromatography was coupled with reverse-phase HPLC to follow the formation of complexes between Cd and g-glutamyl peptides in cells exposed to the metal ion during the growth cycle. Five stable complexes (Cd /PCn n /2 /6), in which peptides of unique chain length chelate Cd by formation of thiolate bonds, were identified. The formation of Cd /PCn complexes in which acid labile sulfide is incorporated (PC-coated CdS crystallites) was also demonstrated. These complexes exhibited luminescence and UV transition at 280 nm. The apparent MW was 8 /12 kDa and the S2 /Cd ratio 0.4. The 66% of the total PC g-Glu /Cys units in these complexes were polymerized as PC4. The incorporation of sulfide into Cd /PCn complexes enhances the Cd/SCys ratio from 0.6 to 1.6. The kinetics of formation of Cd /thiols complexes is consistent with an involvement of the glutathione (GSH) in the Cd sequestration mechanism of P. tricornutum through the initial formation of Cd /GSH complexes. The metal ion is subsequently transferred from GSH to Cd-induced phytochelatins to form more stable Cd /PCn complexes. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadmium; Metal-binding peptides; Phaeodactylum tricornutum ; Phytochelatins; Glutathione
1. Introduction Plants, algae and fungi have developed several defence mechanisms to cope with the occurrence of potentially toxic environmental levels of heavy metals [1] An important mechanism of metal detoxification involves intracellular thiol-containing compounds, such as glutathione (GSH) and phytochelatins (PCn ). PCn are low molecular weight (LMW), cysteine-rich peptides of general structure (g-Glu /Cys)n /Gly, with n generally ranging from 2 to 6. capable to bind metal ions via thiolate coordination. They are synthesized by a specific enzyme, the PC synthase, that is activated by the presence of metal ions and uses GSH, or PCn , as substrate [2 /4]. The formation of metal/PCn complexes in the cell effectively reduces the intracellular concentra-
* Corresponding author. Tel.: /39-050-315-2754; fax: /39-050315-2760 E-mail address: [email protected] (G. Scarano).
tion of the free metal ion by sequestering it into harmless compounds. Studies on the sequestration of metal ions, in particular Cd, in these complexes, and on their characterization, have mainly regarded yeasts and higher plants [5,6], for which two main groups of Cd /PCn complexes have been resolved: the high molecular weight (HMW) and the LMW form. The presence of acid labile sulfide (S2) has been demonstrated in the HMW form, in which peptides with different chain length participate in binding Cd. These complexes contain CdS crystallites coated with (g-Glu /Cys)n /Gly peptides [7 /9]. Only recently, a number of LMW complexes have been resolved as distinct Cd /PCn complexes homogeneous in the chain-length of the peptide, in several plant extracts [10,11] and in the marine diatom Phaeodactylum tricornutum [12]. The LMW Cd /PCn complexes are thought to be the nascent form of the HMW complexes [13,14]. The induction of PCn in phytoplanktonic algae has been widely demonstrated both in laboratory cultures
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 2 1 6 - 9
808
E. Morelli et al. / Plant Science 163 (2002) 807 /813
and in field studies [15 /18], but only few papers have regarded the characterization of metal /PCn complexes in these organisms [12,19,20]. In this work, we investigated the cellular Cd speciation and the kinetics of formation of the Cd /g-glutamyl peptides complexes in cells of P. tricornutum exposed to this metal ion during the growth cycle. An approach based on size-exclusion chromatography with off-line detection of non-protein thiols, by reverse-phase high performance liquid chromatography (RP-HPLC), and of metal ion, by atomic absorption spectrometry (AAS), was used to individuate and characterize the Cd /PCn complexes. We show the role of GSH in the Cd sequestration process and the ability of this microalga to incorporate inorganic sulfide in the metal /PCn complexes.
2. Materials and methods P. tricornutum Bohlin (Bacillariophyceae) was grown in axenic conditions, at 23 8C, using continuous illumination under fluorescent daylight (100 mmol photons per m2 s 1). The growth medium was natural seawater enriched with f/2 [21] at one fifth the normal trace metal concentration and without Cu, sterilized by filtration on 0.2 mm sterile membrane filters. Stock cultures were maintained in batch by inoculating every 7 days into a fresh culture medium. In metal-exposure experiments, the culture medium was spiked with Cd to a final concentration of 1 mM. Calculated volumes of the stock cultures of P. tricornutum were used as inoculum to obtain an initial cell concentration of 3/104 cells per ml. The growth was monitored by counting cells by means of a Thoma chamber under a microscope. Samples of algae were harvested daily by filtration onto 0.8 mm membrane filters (Sartorius), then rinsed extensively with natural seawater. The algae were immediately placed in 2 ml of 50 mM N -[2-hydroxyethyl]piperazine-N ?-[2-etbanesulfonic acid] (HEPES)/50 mM NaCl buffer (pH 7.5), containing 1 mM Tris (2-carboxyethyl) phosphine (TCEP) as antioxidant, then disrupted by sonication (Sonopuls Ultrasonic Homogenizer Bandelin) for 10 min with a repeating duty cycle of 0.3 s. All extraction procedures were carried out in an ice bath. The homogenate was assayed for Cd by AAS using a Perkin/Elmer Spectrophotometer (Model 1100 B) equipped with a graphite furnace (Model HGA 700). The cellular homogenate was centrifuged at 5000 /g for 15 min at 4 8C to remove cellular debris, then the supernatant was filtered through 0.2 mm membrane filter and subjected to the SEC procedure. An aliquot of the filtrate was retained for the determination of nonprotein thiols, by RP /HPLC.
Size exclusion chromatography (SEC) was performed by means of a Hi-Load Superdex 30 preparation grade column (60 /1.6 cm; Pharmacia Biotech), by using 50 mM HEPES/50 mM NaCl pH 7.5, as elution buffer at a flow rate of 1 ml min 1. The SEC system consisted of a Pharmacia Biotech Pump P-50, a Rheodyne model IV-7 injection valve and an UV detector (Uvicord SD, Pharmacia LKB) set at 254 nm. The injection volume was 1 ml and the eluate was collected in 1 ml fractions. Before each injection, the column was cleaned by loading 3 ml of 0.1 M EDTA. g-Glulamyl peptides were determined by RP-HPLC after derivatization with the fluorescent tag monobromobimane (mBrB) [22,23] by following the procedure reported elsewhere [12]. Briefly, after acidification (10 ml, 1.2 M HCl/50 mM diethylenetriaminepentacetic acid (DTPA), 30 min), the sample (100 ml) was spiked with 140 ml of 0.7 mM TCEP in 200 mM 4-(2-hydroxyethyl)piperazine-1-propane-sulfonic acid (HEPPS)/5 mM DTPA, pH 8 2, in order to protect the thiol groups from oxidation, and subjected to derivatization (20 ml of 10 mM mBrB). The reaction was allowed to proceed for 15 min in the dark at 45 8C. Afterwards, 20 ml of 100 mM cysteine were added and, 15 min later, the reaction was stopped by adding 20 ml of 1 M methanesulfonic acid (MSA). The derivatized samples were stored in the dark at /4 8C until HPLC analysis. Blank samples were used to identify the reagent peaks. The bimane derivatives were separated on an Alltech Econosphere C-18 5 mm reverse-phase column (250 / 4.6 mm) and detected at 380 nm excitation and 470 nm emission wavelengths. A linear gradient from 10 to 30% acetonitrile in 0.1% trifluoroacetic acid (TFA), for 50 min, was used. The flow rate was 1 ml min 1. Retention time of phytochelatins was checked as previously reported [12], and their quantification was obtained from the relationship peak area versus concentration of GSH standard solutions. The HPLC system consisted of two Shimadzu LC10AD pumps, a Rheodyne 7725 injection valve equipped with a 100 ml loop, and a RF-10AXL. Shimadzu fluorescence detector. Chromatographic data were processed using CHROMATOPLUS software. Acid labile sulfide was determined by the methylene blue method [7,24]. 250 ml of zinc acetate (2.6% in water) and 50 ml of NaOH (6%) were added to 350 ml of the sample. After 5 min, 250 ml of N ,N? -dimethyl-p phenylene diamine (0.1% solution in 5 M HCl) were added and mixed for 1 min. Finally, 50 ml of FeCl3 (11.5 mM in 0.1 M HCl) and 0.5 ml of water were added and the reaction was allowed to stand for 30 min at room temperature. The absorbance at 670 nm was measured. Solutions of Na2S were used as reference standards. Spectroscopic measurements were carried out on a Jasco UV/visible spectrometer (Model 7850) and on a
E. Morelli et al. / Plant Science 163 (2002) 807 /813
Perkin/Elmer luminescence spectrometer (Model LS50B). All reagents were analytical grade: TCEP was from Molecular Probes; DTPA, GSH, cysteine and HEPES were from Fluka; mBrB, HEPPS, Na2S and g-Glu /Cys (g-EC) were from Sigma; MSA, (CH3COO)2Zn ×/2H2O, N ,N ?-dimethyl-p-phenylene diamine, HCl and HNO3, Suprapur grade, were from Merck; acetonitrile and TFA, HPLC grade, were from Baker; EDTA, FeCl3 and CdCl2 were from Carlo Erba. TCEP and mBrB were prepared daily, the other solutions were prepared weekly and stored in the dark at /4 8C. Water was purified by a Milli-Q system (Millipore). Seawater was collected offshore, in an uncontaminated area, then filtered through 0.45 mm membrane filters (Sartorius) and stored in the dark at /4 8C.
3. Results Cells of P. tricornutum growing in the culture medium at 1 mM Cd concentration, showed a growth rate (m/ 1.7 doubling per day) similar to that of a control culture. The exposed culture was harvested daily during a 7 days period, starting from the early exponential phase till the stationary phase, and assayed for Cd and PCn . Fig. 1 shows that, within the first 2 days of exposure, low levels of Cd (nmol Cd per 1 /109 cells) and PCn were detected in cells, thereafter, Cd sharply increased, and PCn (nmol g-Glu /Cys units per 1 /109 cells) began to accumulate, when the culture approached to the stationary growth phase (3rd and 4th day). The very low cellular Cd content assayed at the end of the 2nd day of exposure could be attributed to the dilution by cell division, that is higher during the early exponential phase. Reverse
Fig. 1. Time course of Cd and total PC g-Glu /Cys accumulation in cells of P. tricornutum growing in culture medium spiked with 1 mM Cd (bar chart). Cell density expressed as cell number per ml ( */9 */). Inset: actual total dissolved Cd concentration in the culture medium.
809
phase HPLC assays performed on cellular extracts revealed the presence of PCn with n value from 2 to 6. After the 4th day, concomitantly to a depletion of the total dissolved Cd concentration in solution (Fig. 1, insert), the Cd content in the cells reached a nearly constant value, nevertheless, the PCn synthesis proceeded during the next period of exposure. The time course of total PCn (g-Glu /Cys units) synthesis was almost linear from the 3rd to 5th day, after which the rate decreased. The time course of individual PCn oligomers formation shows that PC4 are accumulated in the cell at a higher rate than the other peptides. At the 7th day of exposure, the total cellular PC g-Glu /Cys units were polymerized as follows: 43% as PC4, 28% as PC3, 22% as PC2, 6% as PC5 and 1% as PC6. GSH cellular pool did not appreciably change during the PCn synthesis, indicating that, under such exposure conditions, the cells replaced the GSH consumed for PCn synthesis. During the 6th and 7th day of exposure, an increase of the cellular pool of g-EC, suggesting the beginning of degradation of Cd /PCn complexes, was observed. The speciation of total Cd in the cell was investigated by fractionating, by SEC, the cell extracts daily obtained from the exposed culture, and by assaying for Cd and for non-protein thiols the column effluent. The chromatograms obtained from Cd assays, in the period from the 3rd to 7th day of exposure (Fig. 2), show that Cd eluting at the exclusion volume of the column (fr. 36/ 44) was bound to cellular components of very HMW;
Fig. 2. Size exclusion chromatograms, obtained from Cd assays, related to crude extracts of 1/109 cells of P. tricornutum . Cells were grown in the presence of 1 mM Cd and harvested daily from the 3rd day after the inoculum (upper curve) until the 7th day (lower curve) SE column: Hi-Load Superdex 30 (60/1.6 cm; Pharmacia Biotech). Elution buffer: 50 mM HEPES/50 mM NaCl, pH 7.5. Flow rate: 1 ml 1 min 1. Volume of the collected fractions: 1 ml.
810
E. Morelli et al. / Plant Science 163 (2002) 807 /813
these fractions did not contain phytochelatins and their Cd content did not change with exposure time. A significant fraction of the Cd in the cellular extracts of the 4th and 5th day eluted at the LMW zone of the column (fr. 90 /105). RP-HPLC assays (Fig. 3A, 5th day sampling) revealed that PC2, GSH and g-Glu/Cys peptide (g-EC) eluted in these fractions. The PC2 peak maximum was coincident with a shoulder on the leading edge of the Cd peak indicating that a fraction of LMW Cd was bound to this oligomer. GSH co-eluted with the tailing edge of the Cd peak, suggesting that Cd /GSH complexes may be present. At the end of the 7th day of exposure (Fig. 3B), only a low amount of Cd eluted in this zone of the column. Two peaks were observed, a Cd peak coincident with the elution of PC2 and a very low, but distinguishable, Cd peak eluting coincident with the GSH peak. Cd eluting in the medium molecular weight (MMW) zone of the column (fr. 55 /90) forms several peaks. A detailed analysis of PCn in the SEC eluate shows that PCn with n values from 3 to 6 are present, and that each oligomer elutes from the column with a profile characterized by two distinct peaks (Fig. 4, 5th day sampling). The elution time of the first peak (fr. 60/ 70) was similar for all oligomers. The peak merging later, showed an elution time different for each oligomer. Since Cd was associated to all these PCn peaks, this pattern indicates that each oligomer, characterized by its value of n, is involved in two different complexes with Cd. By overlapping the chromatograms obtained from Cd and PCn assays, Cd /PCn complexes accumulated in the cell can be characterized. Cd eluting at fractions 85/ 90 was bound to PC3, indicating the formation of stable Cd /PC3 complexes. Cd peak eluting at higher MW (fr. 72 /84) appears composed by two or more overlapping peaks: its maximum was coincident with the maximum of a well defined peak of PC4, and the shoulder on the leading edge of Cd peak was coincident with the elution of PC5 and PC6 oligopeptides, co-eluting in the fractions 71 /75. It was possible to obtain a better separation of
these complexes by using a higher NaCl concentration in the mobile phase [12]. These results show that Cd forms distinct complexes with each oligomer with n value from 3 to 6. Cd eluting at fractions 60/70 gave an enlarged peak composed by two overlapping peaks and was associated with a mixture of PCn to form Cd complexes with an apparent molecular weight of 8 /12 kDa. The peptide composition of these complexes showed that the highest proportion of total PC g-Glu /Cys units (66%) was polymerized as PC4, followed by PC5 (16%), PC3 (10%), PC6 (6%) and PC2 (2%). Further characterization of the eluate was obtained by performing the analysis of acid labile sulfide in SEC fractions. Acid labile sulfide eluted at the fractions 60/ 70, giving an enlarged peak. This peak was coincident with Cd eluting in the fractions where the mixture of PCn was detected, indicating that PCn were involved in Cd-complexes in which inorganic sulfide is incorporated. The mean value of the molar ratio S2/Cd was 0.4. The concentration of acid labile sulfide was under the detection limit of the method in the fractions containing the other distinct Cd /PCn complexes. UV absorption spectra of fractions containing acid labile sulfide showed a transition near 280 nm (Fig. 5). This UV transition was absent in the fractions containing Cd bound to distinct oligomers differing in chain length, for which only the transition to 254 nm, characteristic of the
Fig. 3. Concentration of Cd (m), PC2 (2), GSH (k) and g-EC (j) in the fractions eluting in the LMW zone of the SE column. SEC of crude extracts from cells harvested at the 5th day (A), and at the 7th day (B), after the inoculum.
Fig. 4. Concentration of Cd, phytochelatins and sulfide in the fractions eluting in the MMW zone of the SE column. SEC of crude extracts from cells harvested at the 5th day after the inoculum. Solid line: UV-chromatogram at 254 nm.
E. Morelli et al. / Plant Science 163 (2002) 807 /813
Fig. 5. Absorption spectra of selected fractions of SEC reported in Fig. 4. ( */), ( / / /) and ( */ */) refer to sulfide-containing fractions 62 /63 64 /66 and 67 /69, respectively. ( */ ×/ ×/ */), ( */ ×/ */) and ( ) refer to fractions 87 /89 (Cd /PC3,), 76 /78 (Cd /PC4) and 72 /74 (mixture of Cd /PC4 6), respectively. Inset: emission spectra of fractions 64 /66 ( */) and 76 /78 ( / /). Excitation wavelength, 290 nm.
Cd /thiolate bond, was observed. Samples containing PCn and sulfide exhibited luminescence. The emission spectra showed that excitation at 290 nm caused emission with lmax at 350 nm (Fig. 5, inset). These optical spectroscopic properties indicate the presence of Cd /sulfide bonds in these complexes [25 /28]. The kinetic analysis of the different Cd species accumulated in the cells during 7 days of exposure (Fig. 6) shows that between the 3rd and 4th day, corresponding to the period in which the highest rate of metal uptake occurs, LMW-Cd compounds were the dominant species of Cd in the cell. Cd /PCn complexes (as the sum of sulfide-free and sulfide-containing PCn complexes) are formed to a minor extent. After the 4th day, the fraction of Cd bound to PC3 6 continuously increases and LMW-Cd concomitantly decreases. At the 7th day, about 70% of Cd still eluting in the LMW zone of the column was identified as Cd /PC2 complexes. Since the total cellular Cd does not increase from 4th to 7th day of exposure, the decrease of LMW-Cd with exposure time suggests that the major fraction of Cd eluting as LMW-Cd is progressively transferred to Cd / PC3 6 complexes. At the end of exposure time, 80% of total Cd eluted from the SEC column was bound to PCn (n /2 /6), the remaining Cd eluting at the exclusion volume of the column. With the increase of the exposure time (see Fig. 2) the Cd peaks related to both Cd /PCn , and CdS-containing PCn complexes were slightly shifted toward shorter elution time, indicating an increase of their apparent molecular weight. A replicate exposure experiment, in which the time course of cellular Cd and PCn concentration was followed and the chromatographic profiles of Cd and PCn at the 4th and 7th day were obtained, confirmed our results.
811
Fig. 6. Time course of formation of LMW-Cd (%) and MMW-Cd (I) species during the experiment, (k) total PC g-Glu /Cys subunits.
4. Discussion Our results show that the marine diatom P. tricornutum is able to sequester Cd in two classes of Cd /PCn complexes. The first class is characterized by Cdcomplexes with peptides of unique length, in which the peptide chelates Cd by formation of thiolate bonds. Five stable complexes (Cd/PCn n /2 /6) can be individuated according to the ability of P. tricornutum to synthesize phytochelatins with n value from 2 to 6. These complexes have an apparent molecular weight according to the polymerization degree of the oligopeptide involved. The second class of Cd /PCn complexes is characterized by a higher apparent molecular weight and differs from the former for its spectroscopic properties. In these complexes, the metal ion is bound to a heterogeneous mixture of peptides with different chain length and to acid labile sulfide to form CdS-containing PC complexes. For their properties, these Cd /PCn complexes are similar to the PC-coated CdS crystallites previously described by other Authors in several plants and yeasts [7 /9,25 /28]. To our knowledge, this is the first report showing the ability of a marine phytoplanktonic alga to sequester Cd in CdS-containing PC complexes. Only a recent work reports these complexes in the freshwater microalga Chlamydomonas reinhardtii [20]. The incorporation of inorganic sulfide into the Cd / PCn complexes of P. tricornutum enhances the Cd/SCys molar ratio from 0.6 to 1.6, in agreement with the finding that Cd-binding stoichiometry increases with the amount of the sulfide in the complex [25,28]. Sulfide-containing Cd /PCn complexes isolated from P. tricornutum showed a S2/ Cd ratio of 0.4 and displayed UV transition at approximately 280 nm. Native isolates of Candida glabrata and Schizosaccharomyces pombe (S2/ Cd ratio of 0.6 /0.7, respectively) exhibited UV transition near 300/315 nm, and con˚ diameter PC-coated CdS crystallites [27]. tained 20 A The sulfide-containing HMW complexes isolated from the microalga C. reinhardtii (S2/Cd ratio of 0.22)
812
E. Morelli et al. / Plant Science 163 (2002) 807 /813
showed UV transition at approximately 260 nm. Thus, the value of the electronic transition of CdS-containing PC complexes isolated from P. tricornutum , agrees with the known dependence of the band gap on the S2/Cd ratio of these complexes [28,29]. The kinetics of the different species of Cd accumulated in the cells of P. tricornutum during the exposure (Fig. 3A, Fig. 6) is consistent with an involvement of the GSH in the Cd sequestration mechanism through the initial formation of Cd /GSH complexes. The metal ion bound to GSH should be transferred to the newly synthesized (g-Glu/Cys)n /Gly oligomers to form more stable Cd /PCn complexes or, alternatively, Cd /GSH complexes should be converted to Cd /PCn complexes by action of the PC-synthase on this substrate [4]. Our results agree with an in vivo study showing that cells of C . glabrata can sequester Cd by forming Cd /GSH complexes evolving with time of the culture to Cd /PCn complexes [30], as well as with in-vitro studies showing than GSH can transfer several metal ions, such as Hg(II) [31], Cu(I) [32], Pb [33] and Cd [29], into phytochelatins. In a previous our study on Cd sequestration by P. tricornutum , performed under different exposure conditions (10 mM Cd, 6 h exposure, cells in the stationary growth phase), the sulfide-containing Cd /PCn complexes were at undetectable level in cellular extract, and only the Cd /PCn complexes homogeneous in the chain length of the peptide were demonstrated [12]. The findings here reported show that P. tricornutum is capable to sequester Cd in stable PC-coated CdS crystallites, in agreement with similar reports for plants and yeasts. Since the incorporation of sulfide into Cd / PCn clusters is the result of a Cd-induced enhancement of intracellular inorganic sulfide [7,34], apparently, under the exposure conditions previously used, Cd does not stimulate the S2 synthesis to a sufficient extent to produce measurable cellular levels of sulfidecontaining Cd /PCn complexes. An explanation could be that Cd added during the early exponential phase of the cells has promoted an enhanced sulfide production. It has been shown that the addition of Cd to cultures of S . pombe during different stages of the growth cycle results in different intracellular inorganic sulfide production [34]. The importance of sulfide incorporation into PCn in Cd-tolerance is supported by several studies. Metal tolerant ecotypes of Silene vulgaris incorporate into metal/PCn complexes a higher amount of sulfide than the sensitive one [35]. Also, Cd-resistant mutant of yeast C. glabrata exhibited an enhanced production of PC-coated CdS crystallites [36], and Cd-sensitive mutants of S. pombe were unable to synthesize PC-coated CdS crystallites [14,37]. The capability of P. tricornutum to incorporate inorganic sulfide into Cd-peptide clusters could represent an important tool for the defense of this marine diatom from heavy metal stress.
Acknowledgements B.H. Cruz thanks Segretaria Nacional de Ciencia, Tecnologia e Innovacion (SENACYT) and Institute para la Formacion y Aprovechamı`ento de Recursos Humanos (IFARHU) from the Republic of Panama for a Ph.D. grant, and Generalitat de Catalunya (Departament d’ Universitats, Recerca i Societat de la Informacio´) for a grant which allowed his stay in Italy.
References [1] J.L. Hall, Cellular mechanisms for heavy metal detoxification and tolerance, J. Exp. Bot. 53 (2002) 1 /11. [2] E. Grill, E.L. Winnacker, M.H. Zenk, Phytochelatins: the principal heavy-metal complexing peptides of higher plants, Science 230 (1985) 674 /676. [3] E. Grill, S. Loffler, E.L. Winnacker, M.H. Zenk, Phytochelatins, the heavy-metal-binding-peptides of plants, are synthesized from glutathione by a specific g-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase), Proc. Natl. Acad. Sci. USA 86 (1989) 6838 /6842. [4] C.S. Cobbett, Phytochelatin biosynthesis and function in heavymetal detoxification, Curr. Opin. Plant Biol. 3 (2000) 211 /216. [5] W.E. Rauser, Phytochelatins and related peptides, structure, biosynthesis and function, Plant Physiol. 109 (1995) 1141 /1149. [6] M.H. Zenk, Heavy metals detoxification in higher plants */a review, Gene 179 (1996) 21 /30. [7] R. Kneer, M.H. Zenk, The formation of Cd-phytochelatin complexes in plant cell cultures, Phytochemistry 44 (1997) 69 /74. [8] W.E. Rauser, Roots of maize seedlings retain most of their cadmium through two complexes, J. Plant Physiol. 156 (2000) 545 /551. [9] D.R. Winge, C.T. Dameron, R.K. Mehra, Metal: sulfide quantum crystallites in yeast, in: M.J. Stillman, C.F. Shaw, III, K.T. Suzuki (Eds.), Metallothioneins, VCH Publishers, New York, 1992, pp. 257 /270. [10] T.-Y. Yen, J.A. Villa, J.G. DeWitt, Analysis of phytochelatin / cadmium complexes from plant tissue culture using nano-electrospray ionization tandem mass spectrometry and capillary liquid chromatography/electrospray ionization tandem mass spectrometry, J. Mass Spectrom. 34 (1999) 930 /941. [11] V. Vacchina, K. Polec, J. Szpunar, Speciation of cadmium in plant tissues by size-exclusion chromatography with ICP-MS detection, J. Anal. At. Spectrom. 14 (1999) 1557 /1566. [12] G. Scarano, E. Morelli, Characterization of cadmium- and lead / phytochelatin complexes formed in a marine microalga in response to metal exposure, BioMetals 15 (2002) 145 /151. [13] D.F. Ortiz, T. Ruscitti, K.F. McCue, D.W. Ow, Transport of metal-binding peptides by HMT1, a fission yeast ABC-type vacuolar membrane protein, J. Biol. Chem. 270 (1995) 4721 / 4728. [14] D.M. Speiser, D.F. Ortiz, L. Kreppel, G. Scheel, G. McDonald, D.W. Ow, Purine biosynthetic genes are required for cadmium tolerance in Schyzosaccharomyces pombe , Mol. Cell. Biol. 12 (1992) 5301 /5310. [15] B.A. Ahner, S. Kong, F.M.M. Morel, Phytochelatin production in marine algae. 1. An interspecies comparison, Limnol. Oceanog. 40 (1995) 649 /657. [16] B.A. Ahner, F.M.M. Morel, Phytochelatin production in marine algae. 2. Induction by various metals, Limnol. Oceanog. 40 (1995) 658 /665.
E. Morelli et al. / Plant Science 163 (2002) 807 /813 [17] B.A. Ahner, F.M.M. Morel, J.W. Moffett, Trace metal control of phytochelatin production in coastal waters, Limnol. Oceanog. 42 (1997) 601 /608. [18] K. Knauer, B.A. Ahner, I.B. Xue, L. Sigg, Metal and phytochelatin content in phytoplankton from freshwater lakes with different metal concentrations, Environ. Toxicol. Chem. 17 (1998) 2444 /2452. [19] W. Gekeler, E. Grill, E.L. Winnacker, M.H. Zenk, Algae sequester heavy metals via synthesis of phytochelatin complexes, Arch. Microbiol. 150 (1988) 197 /202. [20] S. Hu, K.W.K. Law, M. Wu, Cadmium sequestration in Chlamydomonas reinhardtii , Plant Sci. 161 (2001) 987 /996. [21] R.R.L. Guillard, Cultures of phytoplankton for feeding marine invertebrates, in: W.L. Smith, M.H. Chanley (Eds.), Culture of Marine Invertebrates Animals, Plenum Press, New York, 1975, pp. 29 /60. [22] G.L. Newton, L. Dorian, R.C. Fahey, Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography, Anal. Biochem. 114 (1981) 383 /387. [23] R.C. Fahey, G.L. Newton, Determination of low-molecularweight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography, in: W.B. Jakoby, O.W. Griffith (Eds.), Methods in Enzymology, vol. 143, Academic Press, New York, 1987, pp. 85 /96. [24] J.C. Rabinowitz, Analysis of acid-labile sulfide and sulfhydryl groups, in: S. Fleischer, L. Packer (Eds.), Methods in Enzymology, vol. 53, Academic Press, New York, 1978, pp. 275 /277. [25] R.N. Reese, D.R. Winge, Sulfide stabilization of the cadmium /gglutamyl peptide complex of Schyzosaccharomyces pombe , J. Biol. Chem. 263 (1988) 12832 /12835. [26] R.N. Reese, R.K. Mehra, E.B. Tarbet, D.R. Winge, Studies on the g-glutamyl Cu-binding peptide from Schyzosaccharomyces pombe , J. Biol. Chem. 263 (1988) 4186 /4192. [27] C.T. Dameron, R.N. Reese, R.K. Mehra, A.R. Kortan, P.J. Carroll, M.L. Steigerwald, L.E. Brus, D.R. Winge, Biosynthesis
[28] [29]
[30]
[31]
[32] [33]
[34]
[35]
[36]
[37]
813
of cadmium sulphide quantum semiconductor crystallites, Nature 338 (1989) 596 /597. C.T. Dameron, D.R. Winge, Characterization of peptide-coated cadmium-suifide crystallites, Inorg. Chem. 29 (1990) 1343 /1348. W. Bae, R.K. Mehra, Properties of glutathione- and phytochelatin-capped CdS bionanocrystallites, J. Inorg. Biochem. 69 (1998) 33 /43. J. Barbas, W. Santhanagopalan, M. Blaszczynski, W.R. Ellis, Jr, D.R. Winge, Conversion in the peptides coating cadmium:sulfide crystallites in Candida glabrata , J. Inorg. Biochem. 48 (1992) 95 / 105. R.K. Mehra, J. Miclat, W.R. Kodati, R. Abdullah, T.C. Hunter, P. Mulchandani, Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins, Biochem. J. 314 (1996) 73 /82. R.K. Mehra, P. Mulchandani, Glutathione-mediated transfer of Cu(I) into phytochelatins, Biochem. J. 307 (1995) 697 /705. R.K. Mehra, V.R. Kodati, R. Abdullah, Chain length-dependent Pb(II)-coordination in phytochelatins, Biochem. Biophys. Res. Commun. 215 (1995) 730 /736. P. Williams, E. Keshavarz-Moore, P. Dunnill, Production of cadmium sulphide microcrystallites in batch cultivation by Schyzosaccharomyces pombe , J. Biotechnol. 48 (1996) 259 / 267. J.A.C. Verkleij, P. Koevoets, J. Van’t Riet, R. Bark, Y. Mijdam, W.H.O. Ernst, poly(g-Glutamylcysteinyl)-glycines or phytochelatins and their role in cadmium tolerance of Silene vulgaris , Plant Cell Environ. 13 (1990) 913 /921. R.K. Mehra, P. Mulchandani, T.C. Hunter, Role of CdS quantum crystallites in cadmium resistance in Candida glabrata , Biochem. Biophys. Res. Commun. 200 (1994) 1193 /1200. D.F. Ortiz, L. Kreppel, D.M. Speiser, G. Scheel, G. McDonald, D.W. Ow, Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter, EMBO J. 11 (1992) 3491 /3499.
Speciation of cadmium g-glutamyl peptides complexes in cells of the marine microalga Phaeodactylum tricornutum /
Elisabetta Morelli a, Boris H. Cruz b, Simone Somovigo a, Gioacchino Scarano a,* a
Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Area della Ricerca di Pisa, Via G. Moruzzi 1, 56124 Pisa, Italy b Departament de Quimica Analitica, Universitat de Barcelona, Av. Diagonal 647, E 08028 Barcelona, Spain Received 17 May 2002; received in revised form 2 July 2002; accepted 3 July 2002
Abstract Phaeodactylum tricornutum responds to Cd exposure by synthesizing phytochelatins (PCn ) [(g-Glu /Cys)n /Gly] with n values from 2 to 6. Size exclusion chromatography was coupled with reverse-phase HPLC to follow the formation of complexes between Cd and g-glutamyl peptides in cells exposed to the metal ion during the growth cycle. Five stable complexes (Cd /PCn n /2 /6), in which peptides of unique chain length chelate Cd by formation of thiolate bonds, were identified. The formation of Cd /PCn complexes in which acid labile sulfide is incorporated (PC-coated CdS crystallites) was also demonstrated. These complexes exhibited luminescence and UV transition at 280 nm. The apparent MW was 8 /12 kDa and the S2 /Cd ratio 0.4. The 66% of the total PC g-Glu /Cys units in these complexes were polymerized as PC4. The incorporation of sulfide into Cd /PCn complexes enhances the Cd/SCys ratio from 0.6 to 1.6. The kinetics of formation of Cd /thiols complexes is consistent with an involvement of the glutathione (GSH) in the Cd sequestration mechanism of P. tricornutum through the initial formation of Cd /GSH complexes. The metal ion is subsequently transferred from GSH to Cd-induced phytochelatins to form more stable Cd /PCn complexes. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadmium; Metal-binding peptides; Phaeodactylum tricornutum ; Phytochelatins; Glutathione
1. Introduction Plants, algae and fungi have developed several defence mechanisms to cope with the occurrence of potentially toxic environmental levels of heavy metals [1] An important mechanism of metal detoxification involves intracellular thiol-containing compounds, such as glutathione (GSH) and phytochelatins (PCn ). PCn are low molecular weight (LMW), cysteine-rich peptides of general structure (g-Glu /Cys)n /Gly, with n generally ranging from 2 to 6. capable to bind metal ions via thiolate coordination. They are synthesized by a specific enzyme, the PC synthase, that is activated by the presence of metal ions and uses GSH, or PCn , as substrate [2 /4]. The formation of metal/PCn complexes in the cell effectively reduces the intracellular concentra-
* Corresponding author. Tel.: /39-050-315-2754; fax: /39-050315-2760 E-mail address: [email protected] (G. Scarano).
tion of the free metal ion by sequestering it into harmless compounds. Studies on the sequestration of metal ions, in particular Cd, in these complexes, and on their characterization, have mainly regarded yeasts and higher plants [5,6], for which two main groups of Cd /PCn complexes have been resolved: the high molecular weight (HMW) and the LMW form. The presence of acid labile sulfide (S2) has been demonstrated in the HMW form, in which peptides with different chain length participate in binding Cd. These complexes contain CdS crystallites coated with (g-Glu /Cys)n /Gly peptides [7 /9]. Only recently, a number of LMW complexes have been resolved as distinct Cd /PCn complexes homogeneous in the chain-length of the peptide, in several plant extracts [10,11] and in the marine diatom Phaeodactylum tricornutum [12]. The LMW Cd /PCn complexes are thought to be the nascent form of the HMW complexes [13,14]. The induction of PCn in phytoplanktonic algae has been widely demonstrated both in laboratory cultures
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 2 1 6 - 9
808
E. Morelli et al. / Plant Science 163 (2002) 807 /813
and in field studies [15 /18], but only few papers have regarded the characterization of metal /PCn complexes in these organisms [12,19,20]. In this work, we investigated the cellular Cd speciation and the kinetics of formation of the Cd /g-glutamyl peptides complexes in cells of P. tricornutum exposed to this metal ion during the growth cycle. An approach based on size-exclusion chromatography with off-line detection of non-protein thiols, by reverse-phase high performance liquid chromatography (RP-HPLC), and of metal ion, by atomic absorption spectrometry (AAS), was used to individuate and characterize the Cd /PCn complexes. We show the role of GSH in the Cd sequestration process and the ability of this microalga to incorporate inorganic sulfide in the metal /PCn complexes.
2. Materials and methods P. tricornutum Bohlin (Bacillariophyceae) was grown in axenic conditions, at 23 8C, using continuous illumination under fluorescent daylight (100 mmol photons per m2 s 1). The growth medium was natural seawater enriched with f/2 [21] at one fifth the normal trace metal concentration and without Cu, sterilized by filtration on 0.2 mm sterile membrane filters. Stock cultures were maintained in batch by inoculating every 7 days into a fresh culture medium. In metal-exposure experiments, the culture medium was spiked with Cd to a final concentration of 1 mM. Calculated volumes of the stock cultures of P. tricornutum were used as inoculum to obtain an initial cell concentration of 3/104 cells per ml. The growth was monitored by counting cells by means of a Thoma chamber under a microscope. Samples of algae were harvested daily by filtration onto 0.8 mm membrane filters (Sartorius), then rinsed extensively with natural seawater. The algae were immediately placed in 2 ml of 50 mM N -[2-hydroxyethyl]piperazine-N ?-[2-etbanesulfonic acid] (HEPES)/50 mM NaCl buffer (pH 7.5), containing 1 mM Tris (2-carboxyethyl) phosphine (TCEP) as antioxidant, then disrupted by sonication (Sonopuls Ultrasonic Homogenizer Bandelin) for 10 min with a repeating duty cycle of 0.3 s. All extraction procedures were carried out in an ice bath. The homogenate was assayed for Cd by AAS using a Perkin/Elmer Spectrophotometer (Model 1100 B) equipped with a graphite furnace (Model HGA 700). The cellular homogenate was centrifuged at 5000 /g for 15 min at 4 8C to remove cellular debris, then the supernatant was filtered through 0.2 mm membrane filter and subjected to the SEC procedure. An aliquot of the filtrate was retained for the determination of nonprotein thiols, by RP /HPLC.
Size exclusion chromatography (SEC) was performed by means of a Hi-Load Superdex 30 preparation grade column (60 /1.6 cm; Pharmacia Biotech), by using 50 mM HEPES/50 mM NaCl pH 7.5, as elution buffer at a flow rate of 1 ml min 1. The SEC system consisted of a Pharmacia Biotech Pump P-50, a Rheodyne model IV-7 injection valve and an UV detector (Uvicord SD, Pharmacia LKB) set at 254 nm. The injection volume was 1 ml and the eluate was collected in 1 ml fractions. Before each injection, the column was cleaned by loading 3 ml of 0.1 M EDTA. g-Glulamyl peptides were determined by RP-HPLC after derivatization with the fluorescent tag monobromobimane (mBrB) [22,23] by following the procedure reported elsewhere [12]. Briefly, after acidification (10 ml, 1.2 M HCl/50 mM diethylenetriaminepentacetic acid (DTPA), 30 min), the sample (100 ml) was spiked with 140 ml of 0.7 mM TCEP in 200 mM 4-(2-hydroxyethyl)piperazine-1-propane-sulfonic acid (HEPPS)/5 mM DTPA, pH 8 2, in order to protect the thiol groups from oxidation, and subjected to derivatization (20 ml of 10 mM mBrB). The reaction was allowed to proceed for 15 min in the dark at 45 8C. Afterwards, 20 ml of 100 mM cysteine were added and, 15 min later, the reaction was stopped by adding 20 ml of 1 M methanesulfonic acid (MSA). The derivatized samples were stored in the dark at /4 8C until HPLC analysis. Blank samples were used to identify the reagent peaks. The bimane derivatives were separated on an Alltech Econosphere C-18 5 mm reverse-phase column (250 / 4.6 mm) and detected at 380 nm excitation and 470 nm emission wavelengths. A linear gradient from 10 to 30% acetonitrile in 0.1% trifluoroacetic acid (TFA), for 50 min, was used. The flow rate was 1 ml min 1. Retention time of phytochelatins was checked as previously reported [12], and their quantification was obtained from the relationship peak area versus concentration of GSH standard solutions. The HPLC system consisted of two Shimadzu LC10AD pumps, a Rheodyne 7725 injection valve equipped with a 100 ml loop, and a RF-10AXL. Shimadzu fluorescence detector. Chromatographic data were processed using CHROMATOPLUS software. Acid labile sulfide was determined by the methylene blue method [7,24]. 250 ml of zinc acetate (2.6% in water) and 50 ml of NaOH (6%) were added to 350 ml of the sample. After 5 min, 250 ml of N ,N? -dimethyl-p phenylene diamine (0.1% solution in 5 M HCl) were added and mixed for 1 min. Finally, 50 ml of FeCl3 (11.5 mM in 0.1 M HCl) and 0.5 ml of water were added and the reaction was allowed to stand for 30 min at room temperature. The absorbance at 670 nm was measured. Solutions of Na2S were used as reference standards. Spectroscopic measurements were carried out on a Jasco UV/visible spectrometer (Model 7850) and on a
E. Morelli et al. / Plant Science 163 (2002) 807 /813
Perkin/Elmer luminescence spectrometer (Model LS50B). All reagents were analytical grade: TCEP was from Molecular Probes; DTPA, GSH, cysteine and HEPES were from Fluka; mBrB, HEPPS, Na2S and g-Glu /Cys (g-EC) were from Sigma; MSA, (CH3COO)2Zn ×/2H2O, N ,N ?-dimethyl-p-phenylene diamine, HCl and HNO3, Suprapur grade, were from Merck; acetonitrile and TFA, HPLC grade, were from Baker; EDTA, FeCl3 and CdCl2 were from Carlo Erba. TCEP and mBrB were prepared daily, the other solutions were prepared weekly and stored in the dark at /4 8C. Water was purified by a Milli-Q system (Millipore). Seawater was collected offshore, in an uncontaminated area, then filtered through 0.45 mm membrane filters (Sartorius) and stored in the dark at /4 8C.
3. Results Cells of P. tricornutum growing in the culture medium at 1 mM Cd concentration, showed a growth rate (m/ 1.7 doubling per day) similar to that of a control culture. The exposed culture was harvested daily during a 7 days period, starting from the early exponential phase till the stationary phase, and assayed for Cd and PCn . Fig. 1 shows that, within the first 2 days of exposure, low levels of Cd (nmol Cd per 1 /109 cells) and PCn were detected in cells, thereafter, Cd sharply increased, and PCn (nmol g-Glu /Cys units per 1 /109 cells) began to accumulate, when the culture approached to the stationary growth phase (3rd and 4th day). The very low cellular Cd content assayed at the end of the 2nd day of exposure could be attributed to the dilution by cell division, that is higher during the early exponential phase. Reverse
Fig. 1. Time course of Cd and total PC g-Glu /Cys accumulation in cells of P. tricornutum growing in culture medium spiked with 1 mM Cd (bar chart). Cell density expressed as cell number per ml ( */9 */). Inset: actual total dissolved Cd concentration in the culture medium.
809
phase HPLC assays performed on cellular extracts revealed the presence of PCn with n value from 2 to 6. After the 4th day, concomitantly to a depletion of the total dissolved Cd concentration in solution (Fig. 1, insert), the Cd content in the cells reached a nearly constant value, nevertheless, the PCn synthesis proceeded during the next period of exposure. The time course of total PCn (g-Glu /Cys units) synthesis was almost linear from the 3rd to 5th day, after which the rate decreased. The time course of individual PCn oligomers formation shows that PC4 are accumulated in the cell at a higher rate than the other peptides. At the 7th day of exposure, the total cellular PC g-Glu /Cys units were polymerized as follows: 43% as PC4, 28% as PC3, 22% as PC2, 6% as PC5 and 1% as PC6. GSH cellular pool did not appreciably change during the PCn synthesis, indicating that, under such exposure conditions, the cells replaced the GSH consumed for PCn synthesis. During the 6th and 7th day of exposure, an increase of the cellular pool of g-EC, suggesting the beginning of degradation of Cd /PCn complexes, was observed. The speciation of total Cd in the cell was investigated by fractionating, by SEC, the cell extracts daily obtained from the exposed culture, and by assaying for Cd and for non-protein thiols the column effluent. The chromatograms obtained from Cd assays, in the period from the 3rd to 7th day of exposure (Fig. 2), show that Cd eluting at the exclusion volume of the column (fr. 36/ 44) was bound to cellular components of very HMW;
Fig. 2. Size exclusion chromatograms, obtained from Cd assays, related to crude extracts of 1/109 cells of P. tricornutum . Cells were grown in the presence of 1 mM Cd and harvested daily from the 3rd day after the inoculum (upper curve) until the 7th day (lower curve) SE column: Hi-Load Superdex 30 (60/1.6 cm; Pharmacia Biotech). Elution buffer: 50 mM HEPES/50 mM NaCl, pH 7.5. Flow rate: 1 ml 1 min 1. Volume of the collected fractions: 1 ml.
810
E. Morelli et al. / Plant Science 163 (2002) 807 /813
these fractions did not contain phytochelatins and their Cd content did not change with exposure time. A significant fraction of the Cd in the cellular extracts of the 4th and 5th day eluted at the LMW zone of the column (fr. 90 /105). RP-HPLC assays (Fig. 3A, 5th day sampling) revealed that PC2, GSH and g-Glu/Cys peptide (g-EC) eluted in these fractions. The PC2 peak maximum was coincident with a shoulder on the leading edge of the Cd peak indicating that a fraction of LMW Cd was bound to this oligomer. GSH co-eluted with the tailing edge of the Cd peak, suggesting that Cd /GSH complexes may be present. At the end of the 7th day of exposure (Fig. 3B), only a low amount of Cd eluted in this zone of the column. Two peaks were observed, a Cd peak coincident with the elution of PC2 and a very low, but distinguishable, Cd peak eluting coincident with the GSH peak. Cd eluting in the medium molecular weight (MMW) zone of the column (fr. 55 /90) forms several peaks. A detailed analysis of PCn in the SEC eluate shows that PCn with n values from 3 to 6 are present, and that each oligomer elutes from the column with a profile characterized by two distinct peaks (Fig. 4, 5th day sampling). The elution time of the first peak (fr. 60/ 70) was similar for all oligomers. The peak merging later, showed an elution time different for each oligomer. Since Cd was associated to all these PCn peaks, this pattern indicates that each oligomer, characterized by its value of n, is involved in two different complexes with Cd. By overlapping the chromatograms obtained from Cd and PCn assays, Cd /PCn complexes accumulated in the cell can be characterized. Cd eluting at fractions 85/ 90 was bound to PC3, indicating the formation of stable Cd /PC3 complexes. Cd peak eluting at higher MW (fr. 72 /84) appears composed by two or more overlapping peaks: its maximum was coincident with the maximum of a well defined peak of PC4, and the shoulder on the leading edge of Cd peak was coincident with the elution of PC5 and PC6 oligopeptides, co-eluting in the fractions 71 /75. It was possible to obtain a better separation of
these complexes by using a higher NaCl concentration in the mobile phase [12]. These results show that Cd forms distinct complexes with each oligomer with n value from 3 to 6. Cd eluting at fractions 60/70 gave an enlarged peak composed by two overlapping peaks and was associated with a mixture of PCn to form Cd complexes with an apparent molecular weight of 8 /12 kDa. The peptide composition of these complexes showed that the highest proportion of total PC g-Glu /Cys units (66%) was polymerized as PC4, followed by PC5 (16%), PC3 (10%), PC6 (6%) and PC2 (2%). Further characterization of the eluate was obtained by performing the analysis of acid labile sulfide in SEC fractions. Acid labile sulfide eluted at the fractions 60/ 70, giving an enlarged peak. This peak was coincident with Cd eluting in the fractions where the mixture of PCn was detected, indicating that PCn were involved in Cd-complexes in which inorganic sulfide is incorporated. The mean value of the molar ratio S2/Cd was 0.4. The concentration of acid labile sulfide was under the detection limit of the method in the fractions containing the other distinct Cd /PCn complexes. UV absorption spectra of fractions containing acid labile sulfide showed a transition near 280 nm (Fig. 5). This UV transition was absent in the fractions containing Cd bound to distinct oligomers differing in chain length, for which only the transition to 254 nm, characteristic of the
Fig. 3. Concentration of Cd (m), PC2 (2), GSH (k) and g-EC (j) in the fractions eluting in the LMW zone of the SE column. SEC of crude extracts from cells harvested at the 5th day (A), and at the 7th day (B), after the inoculum.
Fig. 4. Concentration of Cd, phytochelatins and sulfide in the fractions eluting in the MMW zone of the SE column. SEC of crude extracts from cells harvested at the 5th day after the inoculum. Solid line: UV-chromatogram at 254 nm.
E. Morelli et al. / Plant Science 163 (2002) 807 /813
Fig. 5. Absorption spectra of selected fractions of SEC reported in Fig. 4. ( */), ( / / /) and ( */ */) refer to sulfide-containing fractions 62 /63 64 /66 and 67 /69, respectively. ( */ ×/ ×/ */), ( */ ×/ */) and ( ) refer to fractions 87 /89 (Cd /PC3,), 76 /78 (Cd /PC4) and 72 /74 (mixture of Cd /PC4 6), respectively. Inset: emission spectra of fractions 64 /66 ( */) and 76 /78 ( / /). Excitation wavelength, 290 nm.
Cd /thiolate bond, was observed. Samples containing PCn and sulfide exhibited luminescence. The emission spectra showed that excitation at 290 nm caused emission with lmax at 350 nm (Fig. 5, inset). These optical spectroscopic properties indicate the presence of Cd /sulfide bonds in these complexes [25 /28]. The kinetic analysis of the different Cd species accumulated in the cells during 7 days of exposure (Fig. 6) shows that between the 3rd and 4th day, corresponding to the period in which the highest rate of metal uptake occurs, LMW-Cd compounds were the dominant species of Cd in the cell. Cd /PCn complexes (as the sum of sulfide-free and sulfide-containing PCn complexes) are formed to a minor extent. After the 4th day, the fraction of Cd bound to PC3 6 continuously increases and LMW-Cd concomitantly decreases. At the 7th day, about 70% of Cd still eluting in the LMW zone of the column was identified as Cd /PC2 complexes. Since the total cellular Cd does not increase from 4th to 7th day of exposure, the decrease of LMW-Cd with exposure time suggests that the major fraction of Cd eluting as LMW-Cd is progressively transferred to Cd / PC3 6 complexes. At the end of exposure time, 80% of total Cd eluted from the SEC column was bound to PCn (n /2 /6), the remaining Cd eluting at the exclusion volume of the column. With the increase of the exposure time (see Fig. 2) the Cd peaks related to both Cd /PCn , and CdS-containing PCn complexes were slightly shifted toward shorter elution time, indicating an increase of their apparent molecular weight. A replicate exposure experiment, in which the time course of cellular Cd and PCn concentration was followed and the chromatographic profiles of Cd and PCn at the 4th and 7th day were obtained, confirmed our results.
811
Fig. 6. Time course of formation of LMW-Cd (%) and MMW-Cd (I) species during the experiment, (k) total PC g-Glu /Cys subunits.
4. Discussion Our results show that the marine diatom P. tricornutum is able to sequester Cd in two classes of Cd /PCn complexes. The first class is characterized by Cdcomplexes with peptides of unique length, in which the peptide chelates Cd by formation of thiolate bonds. Five stable complexes (Cd/PCn n /2 /6) can be individuated according to the ability of P. tricornutum to synthesize phytochelatins with n value from 2 to 6. These complexes have an apparent molecular weight according to the polymerization degree of the oligopeptide involved. The second class of Cd /PCn complexes is characterized by a higher apparent molecular weight and differs from the former for its spectroscopic properties. In these complexes, the metal ion is bound to a heterogeneous mixture of peptides with different chain length and to acid labile sulfide to form CdS-containing PC complexes. For their properties, these Cd /PCn complexes are similar to the PC-coated CdS crystallites previously described by other Authors in several plants and yeasts [7 /9,25 /28]. To our knowledge, this is the first report showing the ability of a marine phytoplanktonic alga to sequester Cd in CdS-containing PC complexes. Only a recent work reports these complexes in the freshwater microalga Chlamydomonas reinhardtii [20]. The incorporation of inorganic sulfide into the Cd / PCn complexes of P. tricornutum enhances the Cd/SCys molar ratio from 0.6 to 1.6, in agreement with the finding that Cd-binding stoichiometry increases with the amount of the sulfide in the complex [25,28]. Sulfide-containing Cd /PCn complexes isolated from P. tricornutum showed a S2/ Cd ratio of 0.4 and displayed UV transition at approximately 280 nm. Native isolates of Candida glabrata and Schizosaccharomyces pombe (S2/ Cd ratio of 0.6 /0.7, respectively) exhibited UV transition near 300/315 nm, and con˚ diameter PC-coated CdS crystallites [27]. tained 20 A The sulfide-containing HMW complexes isolated from the microalga C. reinhardtii (S2/Cd ratio of 0.22)
812
E. Morelli et al. / Plant Science 163 (2002) 807 /813
showed UV transition at approximately 260 nm. Thus, the value of the electronic transition of CdS-containing PC complexes isolated from P. tricornutum , agrees with the known dependence of the band gap on the S2/Cd ratio of these complexes [28,29]. The kinetics of the different species of Cd accumulated in the cells of P. tricornutum during the exposure (Fig. 3A, Fig. 6) is consistent with an involvement of the GSH in the Cd sequestration mechanism through the initial formation of Cd /GSH complexes. The metal ion bound to GSH should be transferred to the newly synthesized (g-Glu/Cys)n /Gly oligomers to form more stable Cd /PCn complexes or, alternatively, Cd /GSH complexes should be converted to Cd /PCn complexes by action of the PC-synthase on this substrate [4]. Our results agree with an in vivo study showing that cells of C . glabrata can sequester Cd by forming Cd /GSH complexes evolving with time of the culture to Cd /PCn complexes [30], as well as with in-vitro studies showing than GSH can transfer several metal ions, such as Hg(II) [31], Cu(I) [32], Pb [33] and Cd [29], into phytochelatins. In a previous our study on Cd sequestration by P. tricornutum , performed under different exposure conditions (10 mM Cd, 6 h exposure, cells in the stationary growth phase), the sulfide-containing Cd /PCn complexes were at undetectable level in cellular extract, and only the Cd /PCn complexes homogeneous in the chain length of the peptide were demonstrated [12]. The findings here reported show that P. tricornutum is capable to sequester Cd in stable PC-coated CdS crystallites, in agreement with similar reports for plants and yeasts. Since the incorporation of sulfide into Cd / PCn clusters is the result of a Cd-induced enhancement of intracellular inorganic sulfide [7,34], apparently, under the exposure conditions previously used, Cd does not stimulate the S2 synthesis to a sufficient extent to produce measurable cellular levels of sulfidecontaining Cd /PCn complexes. An explanation could be that Cd added during the early exponential phase of the cells has promoted an enhanced sulfide production. It has been shown that the addition of Cd to cultures of S . pombe during different stages of the growth cycle results in different intracellular inorganic sulfide production [34]. The importance of sulfide incorporation into PCn in Cd-tolerance is supported by several studies. Metal tolerant ecotypes of Silene vulgaris incorporate into metal/PCn complexes a higher amount of sulfide than the sensitive one [35]. Also, Cd-resistant mutant of yeast C. glabrata exhibited an enhanced production of PC-coated CdS crystallites [36], and Cd-sensitive mutants of S. pombe were unable to synthesize PC-coated CdS crystallites [14,37]. The capability of P. tricornutum to incorporate inorganic sulfide into Cd-peptide clusters could represent an important tool for the defense of this marine diatom from heavy metal stress.
Acknowledgements B.H. Cruz thanks Segretaria Nacional de Ciencia, Tecnologia e Innovacion (SENACYT) and Institute para la Formacion y Aprovechamı`ento de Recursos Humanos (IFARHU) from the Republic of Panama for a Ph.D. grant, and Generalitat de Catalunya (Departament d’ Universitats, Recerca i Societat de la Informacio´) for a grant which allowed his stay in Italy.
References [1] J.L. Hall, Cellular mechanisms for heavy metal detoxification and tolerance, J. Exp. Bot. 53 (2002) 1 /11. [2] E. Grill, E.L. Winnacker, M.H. Zenk, Phytochelatins: the principal heavy-metal complexing peptides of higher plants, Science 230 (1985) 674 /676. [3] E. Grill, S. Loffler, E.L. Winnacker, M.H. Zenk, Phytochelatins, the heavy-metal-binding-peptides of plants, are synthesized from glutathione by a specific g-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase), Proc. Natl. Acad. Sci. USA 86 (1989) 6838 /6842. [4] C.S. Cobbett, Phytochelatin biosynthesis and function in heavymetal detoxification, Curr. Opin. Plant Biol. 3 (2000) 211 /216. [5] W.E. Rauser, Phytochelatins and related peptides, structure, biosynthesis and function, Plant Physiol. 109 (1995) 1141 /1149. [6] M.H. Zenk, Heavy metals detoxification in higher plants */a review, Gene 179 (1996) 21 /30. [7] R. Kneer, M.H. Zenk, The formation of Cd-phytochelatin complexes in plant cell cultures, Phytochemistry 44 (1997) 69 /74. [8] W.E. Rauser, Roots of maize seedlings retain most of their cadmium through two complexes, J. Plant Physiol. 156 (2000) 545 /551. [9] D.R. Winge, C.T. Dameron, R.K. Mehra, Metal: sulfide quantum crystallites in yeast, in: M.J. Stillman, C.F. Shaw, III, K.T. Suzuki (Eds.), Metallothioneins, VCH Publishers, New York, 1992, pp. 257 /270. [10] T.-Y. Yen, J.A. Villa, J.G. DeWitt, Analysis of phytochelatin / cadmium complexes from plant tissue culture using nano-electrospray ionization tandem mass spectrometry and capillary liquid chromatography/electrospray ionization tandem mass spectrometry, J. Mass Spectrom. 34 (1999) 930 /941. [11] V. Vacchina, K. Polec, J. Szpunar, Speciation of cadmium in plant tissues by size-exclusion chromatography with ICP-MS detection, J. Anal. At. Spectrom. 14 (1999) 1557 /1566. [12] G. Scarano, E. Morelli, Characterization of cadmium- and lead / phytochelatin complexes formed in a marine microalga in response to metal exposure, BioMetals 15 (2002) 145 /151. [13] D.F. Ortiz, T. Ruscitti, K.F. McCue, D.W. Ow, Transport of metal-binding peptides by HMT1, a fission yeast ABC-type vacuolar membrane protein, J. Biol. Chem. 270 (1995) 4721 / 4728. [14] D.M. Speiser, D.F. Ortiz, L. Kreppel, G. Scheel, G. McDonald, D.W. Ow, Purine biosynthetic genes are required for cadmium tolerance in Schyzosaccharomyces pombe , Mol. Cell. Biol. 12 (1992) 5301 /5310. [15] B.A. Ahner, S. Kong, F.M.M. Morel, Phytochelatin production in marine algae. 1. An interspecies comparison, Limnol. Oceanog. 40 (1995) 649 /657. [16] B.A. Ahner, F.M.M. Morel, Phytochelatin production in marine algae. 2. Induction by various metals, Limnol. Oceanog. 40 (1995) 658 /665.
E. Morelli et al. / Plant Science 163 (2002) 807 /813 [17] B.A. Ahner, F.M.M. Morel, J.W. Moffett, Trace metal control of phytochelatin production in coastal waters, Limnol. Oceanog. 42 (1997) 601 /608. [18] K. Knauer, B.A. Ahner, I.B. Xue, L. Sigg, Metal and phytochelatin content in phytoplankton from freshwater lakes with different metal concentrations, Environ. Toxicol. Chem. 17 (1998) 2444 /2452. [19] W. Gekeler, E. Grill, E.L. Winnacker, M.H. Zenk, Algae sequester heavy metals via synthesis of phytochelatin complexes, Arch. Microbiol. 150 (1988) 197 /202. [20] S. Hu, K.W.K. Law, M. Wu, Cadmium sequestration in Chlamydomonas reinhardtii , Plant Sci. 161 (2001) 987 /996. [21] R.R.L. Guillard, Cultures of phytoplankton for feeding marine invertebrates, in: W.L. Smith, M.H. Chanley (Eds.), Culture of Marine Invertebrates Animals, Plenum Press, New York, 1975, pp. 29 /60. [22] G.L. Newton, L. Dorian, R.C. Fahey, Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography, Anal. Biochem. 114 (1981) 383 /387. [23] R.C. Fahey, G.L. Newton, Determination of low-molecularweight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography, in: W.B. Jakoby, O.W. Griffith (Eds.), Methods in Enzymology, vol. 143, Academic Press, New York, 1987, pp. 85 /96. [24] J.C. Rabinowitz, Analysis of acid-labile sulfide and sulfhydryl groups, in: S. Fleischer, L. Packer (Eds.), Methods in Enzymology, vol. 53, Academic Press, New York, 1978, pp. 275 /277. [25] R.N. Reese, D.R. Winge, Sulfide stabilization of the cadmium /gglutamyl peptide complex of Schyzosaccharomyces pombe , J. Biol. Chem. 263 (1988) 12832 /12835. [26] R.N. Reese, R.K. Mehra, E.B. Tarbet, D.R. Winge, Studies on the g-glutamyl Cu-binding peptide from Schyzosaccharomyces pombe , J. Biol. Chem. 263 (1988) 4186 /4192. [27] C.T. Dameron, R.N. Reese, R.K. Mehra, A.R. Kortan, P.J. Carroll, M.L. Steigerwald, L.E. Brus, D.R. Winge, Biosynthesis
[28] [29]
[30]
[31]
[32] [33]
[34]
[35]
[36]
[37]
813
of cadmium sulphide quantum semiconductor crystallites, Nature 338 (1989) 596 /597. C.T. Dameron, D.R. Winge, Characterization of peptide-coated cadmium-suifide crystallites, Inorg. Chem. 29 (1990) 1343 /1348. W. Bae, R.K. Mehra, Properties of glutathione- and phytochelatin-capped CdS bionanocrystallites, J. Inorg. Biochem. 69 (1998) 33 /43. J. Barbas, W. Santhanagopalan, M. Blaszczynski, W.R. Ellis, Jr, D.R. Winge, Conversion in the peptides coating cadmium:sulfide crystallites in Candida glabrata , J. Inorg. Biochem. 48 (1992) 95 / 105. R.K. Mehra, J. Miclat, W.R. Kodati, R. Abdullah, T.C. Hunter, P. Mulchandani, Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins, Biochem. J. 314 (1996) 73 /82. R.K. Mehra, P. Mulchandani, Glutathione-mediated transfer of Cu(I) into phytochelatins, Biochem. J. 307 (1995) 697 /705. R.K. Mehra, V.R. Kodati, R. Abdullah, Chain length-dependent Pb(II)-coordination in phytochelatins, Biochem. Biophys. Res. Commun. 215 (1995) 730 /736. P. Williams, E. Keshavarz-Moore, P. Dunnill, Production of cadmium sulphide microcrystallites in batch cultivation by Schyzosaccharomyces pombe , J. Biotechnol. 48 (1996) 259 / 267. J.A.C. Verkleij, P. Koevoets, J. Van’t Riet, R. Bark, Y. Mijdam, W.H.O. Ernst, poly(g-Glutamylcysteinyl)-glycines or phytochelatins and their role in cadmium tolerance of Silene vulgaris , Plant Cell Environ. 13 (1990) 913 /921. R.K. Mehra, P. Mulchandani, T.C. Hunter, Role of CdS quantum crystallites in cadmium resistance in Candida glabrata , Biochem. Biophys. Res. Commun. 200 (1994) 1193 /1200. D.F. Ortiz, L. Kreppel, D.M. Speiser, G. Scheel, G. McDonald, D.W. Ow, Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter, EMBO J. 11 (1992) 3491 /3499.