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assimilation processes
Aquatic Toxicology 84 (2007) 457–464
Sulfur starvation and chromium tolerance in Scenedesmus acutus: A possible link between metal tolerance and the regulation of sulfur uptake/assimilation processes Gessica Gorbi a , Corrado Zanni b , Maria Grazia Corradi b,∗ a
b
Department of Environmental Sciences, University of Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy Department of Evolutionary and Functional Biology, University of Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy Received 2 May 2007; received in revised form 11 July 2007; accepted 17 July 2007
Abstract In a laboratory-selected Cr-tolerant strain of the unicellular green alga Scenedesmus acutus, the capacity to synthesize higher amounts of cysteine (Cys) and reduced glutathione (GSH) than the wild-type was demonstrated to underlie tolerance to Cd and Cr(VI). In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys, hence it was hypothesized that the sensitivity of the two strains to Cr(VI) could be modified after culturing in sulfate-deprived medium. Both strains were grown in the presence of different concentrations or in the absence of sulfate (sulfur-starved) and then assayed for Cr(VI) tolerance in standard medium. Unstarved, sulfur-starved and sulfur-replete cells (cells maintained in standard medium after S-starvation) were analysed for Cys, GSH and sulfur content. Sulfur-starved cells showed a greater tolerance to Cr(VI) than unstarved ones. The increased tolerance was ascribable to a transient physiological change and can be considered as specifically due to sulfur deprivation, since it was lost after a 3-day culture in standard medium and was not exhibited by nitrogen-starved cells. The comparison between Cys, GSH and sulfur content in sulfur-starved and sulfur-replete cells of the two strains suggests that the higher tolerance to Cr(VI) after S-starvation could depend on the up-regulation of sulfate uptake mechanisms, and that the primary reason for the higher tolerance to chromium in the selected strain could be due to greater sensitivity to the decrease in negative intracellular end-products (free Cys and GSH) leading to an earlier up-regulation of sulfate assimilation processes. © 2007 Elsevier B.V. All rights reserved. Keywords: Cr tolerance; Sulfur starvation; Free cysteine; Glutathione; Sulfur content; Scenedesmus acutus
1. Introduction Among the mechanisms involved in heavy metal detoxification in algae, synthesis of reduced glutathione (GSH) and phytochelatins (PCs) plays an important role (Gekeler et al., 1988; Gaur and Rai, 2001; Pawlik-Skowro´nska and Skowro´nski, 2001; Sanit`a di Toppi et al., 2003). Although various studies have focused on this topic, the role of thiol peptides in tolerance to heavy metals in algae is not fully recognised. Torres et al. (1997) ascribed the high tolerance of Cd in Phaeodactylum tricornutum to an increased production of PCs having higher molecular weight compared to other algal species. In addition, Perez-Rama et al. (2001) suggested that the high tolerance to Cd in Tetraselmis suecica could be due to its ability
∗
Corresponding author. Tel.: +39 0521 905597; fax: +39 0521 905403. E-mail address: [email protected] (M.G. Corradi).
0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2007.07.006
to synthesize longer PCs than other algal species. Hu et al. (2001) observed that a Cd-resistant strain of Chlamydomonas reinhardtii produced and maintained significantly higher levels of free Cys than the sensitive strain. The authors suggested that Cys synthesis/turnover and production of high molecular weight (HMW) PC–metal complexes can be linked. Rauser (1999) reported that HMW-complexes contain acid labile sulfide (S2− ) which gives them increased stability and higher metalbinding capacity, as suggested also by Reese et al. (1988) and Reese and Winge (1988). Speiser and Abrahamson (1992) proposed Cys sulfinate as a source of sulfide to be incorporated in PC–metal HMW complex. Hence, besides being the structural thiol aminoacid of GSH and PCs, Cys seems to have an additional role in tolerance to Cd. According to Pawlik-Skowro´nska (2003a,b) the tolerance to high concentrations of zinc in a Zn-tolerant ecotype of the filamentous green alga Stigeoclonium tenue relies on the complexation of metal with Cys-rich derivatives of phytochelatins, novel phytochelatin-related com-
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pounds produced in larger amounts than in the sensitive ecotype. In previous studies, two strains of the unicellular green alga Scenedesmus acutus, namely a wild-type and a laboratoryselected strain tolerant to Cr(VI), Cu and Cd, were taken as a model for the analysis of the mechanisms involved in metal detoxification and tolerance in algae (Corradi et al., 1995a,b, 1998; Gorbi et al., 2002; Torricelli et al., 2004). The tolerant strain showed higher constitutive levels of free Cys than the wildtype strain and significantly higher levels of Cys, GSH and PCs after treatment with Cd (Torricelli et al., 2004). In the tolerant strain, Cys content increased with the increase of Cd concentration and reached a level 10-fold higher than the constitutive one. These results suggested that the ability of the tolerant strain to synthesize high amount of Cys may be a key mechanism of defence against Cd toxicity. Further studies (Gorbi et al., 2006) demonstrated that when subjected to Cr(VI) treatment, the tolerant strain is able to maintain higher GSH levels than the wild type. The comparison of the recovery capacity and GSH levels in the two strains after Cr treatment in sulfate-enriched and -deprived condition clearly demonstrated that sulfate availability influences survival and suggested that tolerance to Cr(VI) is related to GSH biosynthetic capacity. In photosynthetic organisms, sulfate constitutes the main source of sulfur for the biosynthesis of cysteine. In green algae, Cys synthase is regulated; however, Cys increase can be detected only after prolonged sulfur starvation probably because it can be supplied by degradation of glutathione (Schmidt and Jager, 1992). Algae possess an active transport system for sulfate, whose uptake increases after prolonged sulfur starvation (Raven, 1980). Pankow and Janauer (1974) suggested that Cr(VI) toxicity might be due to chromate (CrO4 2− ) anion interfering with the uptake of sulfate (SO4 2− ) anion, which has similar size. Skeffington et al. (1976) reported that sulfate and chromate uptake appears to rely on the same transport system in Hordeum vulgare. Riedel (1985) observed that Cr(VI) uptake in Thalassiosira pseudonana was inversely proportional to external sulfate and Cr(VI) concentrations inhibiting growth also inhibited sulfate uptake. According to Riedel (1985), the result of the competition between sulfate and chromate for uptake is that increasing sulfate concentration promotes increased tolerance and decreased uptake of hexavalent chromium for phytoplankton at least. Deane-Drummond (1987) and Clarkson and Saker (1989) observed that sulfate uptake increased in sulfate-deprived plants and a new or modified sulfate transport system having a higher affinity for sulfate was induced. Yildiz et al. (1994) characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfursufficient and sulfur-deficient conditions and observed that sulfur-deprived cells synthesized a new, high-affinity sulfate transport system which accumulated rapidly and was detectable in cells within 1 h of sulfur deprivation. Kleiman and Cogliatti (1997) reported that when the sulfate transport system is derepressed by a previous sulfate-deprivation, a higher chromate uptake in the presence of sulfate can be observed in Triticum aestivum exposed to high Cr(VI) concentrations. Besides, sulfate uptake in Chlamydomonas reinhardtii and Monoraphidium
braunii is inhibited by anions structurally similar to sulfate such as chromate, selenate and molybdate (P´erez-Casti˜neira et al., 1998). Nocito et al. (2002) observed a 100% increase in sulfate uptake, associated with a decrease in sulfate and GSH content and synthesis of large amounts of PCs in Cd-treated maize roots. These studies indicate that the amount of sulfur available can be a factor influencing the response of algae to chromium. The aim of the present study is to evaluate if the sensitivity of the wild-type and the Cr-tolerant strains of Scenedesmus acutus to hexavalent chromium is modified by culturing in sulfur-limited or sulfur-deprived conditions and to identify which physiological and/or biochemical alterations can be responsible for the differences in sensitivity. The two strains were grown in the presence of different concentrations or in the absence of sulfate, and then treated with Cr(VI) in standard medium. Variations in Cys, GSH and sulfur content in response to sulfur deprivation and repletion were analysed. To evaluate if the response of sulfurstarved algae to chromium can be considered as specifically due to sulfur deprivation, the two strains were also subjected to Cr(VI) treatment after culturing in nitrogen-deprived medium. The primary reason of Cr tolerance in the selected strain is discussed. 2. Materials and methods Culturing of Scenedesmus acutus strains and experiments were carried out in a climate-controlled chamber, at 24 ± 1 ◦ C, 230 mol/m2 s and 16-h photoperiod. Population growth during treatments or recovery was checked by cell counting with a Neubauer haemocytometer by light microscopy. 2.1. Sulfur starvation and Cr tolerance Synchronized axenic stock cultures (initial cell density 3 × 106 cells/mL) of the two strains of Scenedesmus acutus, i.e. the wild-type and the Cr-tolerant strains, were grown for 3 days in 2 L Erlenmeyer flasks in EPA (1971) liquid culture medium modified by lowering zinc concentration to 3.14 g/L. Taking into account the high cell density, salt concentrations were doubled. Aliquots of the stock cultures, in exponential growth phase, were centrifuged for 10 min at 2200 × g and washed with distilled water. The pellets were suspended at 3 × 106 cells/mL density in the standard culture medium (200 mL) or in the medium containing 25, 3 or 0% of the standard sulfate amount (14.36 mg MgSO4 /L). Since MgSO4 is the only source of sulfur in the standard medium, the amount of MgCl2 was simultaneously increased to restore standard magnesium concentration. After 1-, 2-, 4- and 7-day culture, cell density was assessed. After 7-day culture under the different sulfate concentrations, cells of both strains were collected by centrifugation, washed and treated with the lowest Cr(VI) concentration inhibiting growth, i.e. 1 mg/L for the wild-type and 2 mg/L for the Cr-tolerant strain. Treatments with chromium, supplied as K2 Cr2 O7 , were carried out in standard medium (200 mL) at initial cell density of 3 × 106 cells/mL and, after 1, 2, 4 and 7 days of treatment, cell density was checked. The cells cultured in sulfate-deprived medium (0%
G. Gorbi et al. / Aquatic Toxicology 84 (2007) 457–464
S) were treated with 2, 3, 4 or 5 mg Cr(VI)/L as described above. Also, after 7 days of culture in standard medium (unstarved) or in sulfate-deprived medium (S-starved), the cells of both strains were centrifuged, re-suspended in standard fresh medium at 3 × 106 cells/mL density and cultured for 3 days. Afterwards the cells were treated with 1 or 2 mg Cr(VI)/L, depending on the strain, as described above. 2.2. Nitrogen starvation and Cr tolerance Aliquots of the stock cultures of the two strains, in exponential growth phase, were centrifuged for 10 min at 2200 × g and washed with distilled water. The pellets were suspended in standard or in nitrogen-deprived medium to obtain an initial density of 3 × 106 cells/mL and cell density was assessed after 1, 2, 4 and 7 days. Since nitrogen was supplied as NaNO3 , sodium concentration in N-deprived medium was restored by increasing the amount of NaHCO3 appropriately. After the 7-day culture, both strains were treated with 1 and 2 mg Cr(VI)/L in standard medium as described above. 2.3. Dry weight and free cysteine (Cys), reduced glutathione (GSH) and sulfur content Unstarved and S-starved cells of both strains were collected by centrifugation, washed, re-suspended in standard medium at 3 × 106 cells/mL density and cultured for 1, 24 and 48 h at the same conditions as above. From now on, the term “S-replete cells” will be used to indicate S-starved cells which were allowed to recover in standard medium for 1, 24 and 48 h, while the term “S-sufficient cells” will be used to indicate unstarved cells transferred to and maintained in standard medium. Dry weight and free Cys and GSH levels were determined in unstarved, Sstarved, S-replete and S-sufficient cells after 1, 24 and 48 h of recovery, as well as in the cells of the stock cultures. Sulfur content was also determined after 24 h of recovery. The dry weight of the two strains after 7-day culture in N-deprived medium was checked too. For dry weight determination, aliquots (90 mL) of the cultures were filtered on pre-weighed mixed cellulose ester filters with pore size of 0.45 m. The filters were washed three times with doubly distilled water, dried at 95 ◦ C for at least 3 h and weighed. Other aliquots of the same cultures (10 mL) were used to determine cell density by cell counting. Dry weight of 106 cells was calculated. For free Cys and GSH determination, cells were harvested by filtration on a Whatman GF/C filter and extracted using a mortar in ice-cold 5% (w/v) 5-sulfosalicylic acid solution (SSA) with 6.3 mM diethylenetriaminopentaacetic acid (DTPA). After centrifugation at 14,000 × g for 30 min, total non-protein thiol compounds were determined spectrophotometrically at 412 nm in a mixture of 300 L of the supernatants, 1200 L phosphate buffer (pH 7.6) and 25 L Ellman’s reagent. For free Cys determination, 100 L of phosphate buffer were replaced by 0.1 M methyl glyoxal which binds Cys and, after a 10-min reaction, absorbance was determined at the same wave length as above. Cysteine concentration was calculated as the difference between
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total SH and GSH concentration. A calibration curve for standard SH groups (l-Cysteine, MERCK) was used for quantitative determination of Cys and GSH in the extracts. The efficiency of methyl glyoxal in masking Cys was checked on mixtures of Cys and GSH in different proportions. For sulfur determination, 1500 mL of each culture were centrifuged and washed three times with distilled water. The pellet was dried at 95 ◦ C and then turned to powder in a mortar. Subsamples of 15 mg were weighed in tin cups and, after the addition of 10 mg of vanadium pentoxide (V2 O5 ), subjected to controlled combustion at 900 ◦ C. Total sulfur in the combustion products was determined by gas chromatography (carrier gas = He). 2.4. Statistical analysis All experiments were repeated at least three times with the only exception of growth evaluation under different sulfate concentrations. The significance of the differences in growth between the wild-type and the Cr-tolerant strain subjected to Cr treatments after S-starvation was tested by one-way ANOVA after verification of variance homogeneity (Levene’ test). To evaluate the significance of the differences among treatments and between the two strains subjected to the same treatment, dry weight, total sulfur, Cys and GSH data were subjected to one-way ANOVA after verification of variance homogeneity by Levene’ test. Multiple comparison Tukey test was applied when appropriate. 3. Results 3.1. Sulfur starvation and Cr tolerance In the presence of 25% sulfate, the growth of the two strains of Scenedesmus acutus was the same as controls, while it was almost completely inhibited at lower concentration (3%) or in the absence of sulfate (0% S) (Fig. 1). However, after the 7day culture in S-limited (3% S) or -deprived (0% S) medium, the wild-type and the Cr-tolerant strains resumed growth when transferred to standard culture medium (data not shown) and were able to grow even in the presence of Cr(VI) concentrations (i.e. 1 and 2 mg/L, respectively) which inhibited growth of unstarved (100% S) and 25% S cells (Table 1). In addition, after culturing for 7 days in S-deprived medium (S-starvation), the wild-type strain was also able to grow in the presence of 3 mg Cr(VI)/L, reaching a density of approx 5 × 106 cells/mL after a lag phase of 1 day, and the Cr-tolerant strain was not completely inhibited at 4 mg Cr(VI)/L (Fig. 2). Even if able to grow in the presence of Cr(VI) concentration that inhibited unstarved cells, the greater the Cr(VI) concentration, the weaker the growth of S-starved cells (Fig. 2). However, the Cr-tolerant strain reached significantly higher cell densities (p < 0.01) than the wild-type at all Cr(VI) concentrations tested, except for the highest. When allowed to recover for 3 days in standard medium after S-starvation (3 days S-replete), cells of both strains lost the acquired tolerance, undergoing complete growth inhibition in the presence of 1 or 2 mg Cr(VI)/L (Table 1).
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Fig. 1. Growth of the wild-type and Cr-tolerant strains of Scenedesmus acutus in the presence of different concentrations of sulfate (S) expressed as percentage of the standard medium concentration (100% S). Data from a single experiment. Table 1 Cell densities (106 cells/mL) of the wild-type and Cr-tolerant strains after a 7-day treatment with Cr(VI) Strain
Wild-type (1 mg Cr(VI)/L) Cr-tolerant (2 mg Cr(VI)/L)
Pre-cultivation 100% S
25% S
3% S
0% S
3 days S-replete
3.13 (0.20) 3.05 (0.09)
3.06 (0.04) 3.16 (0.04)
8.99 (0.07) 7.97 (0.27)
8.64 (0.44) 9.59 (0.46)
3.07 (0.10) 3.19 (0.04)
Before Cr-treatment, cells of both strains were pre-cultivated as follows: (i) 7 days in standard growth medium (100% S) or in the medium containing 25%, 3% or 0% sulfate (S), (ii) 7 days in S-deprived medium followed by a 3-day recovery in standard medium (3 days S-replete). After pre-cultivation, the cells were collected, suspended in standard medium (3 × 106 cells/mL) supplemented with 1 or 2 mg Cr(VI)/L, depending on the strain. Means of three replicates. In brackets standard deviation.
Fig. 2. Growth of the wild-type and Cr-tolerant strains of Scenedesmus acutus in the presence of various Cr(VI) concentrations after 7 days of pre-cultivation in sulfate-deprived medium. After pre-cultivation the cells were collected, suspended in standard growth medium (3 × 106 cells/mL) and supplemented with different Cr(VI) concentrations. Error bars = standard deviation.
3.2. Nitrogen starvation and Cr tolerance Culturing the cells in the absence of nitrogen completely inhibited cell multiplication in both strains. Following transfer to standard nitrogen conditions, N-starved cells of the wild-type and Cr-tolerant strains started growing immediately and, dur-
ing the first two days, showed a growth rate (4.2 and 3.7 × 106 cells/mL day, respectively) almost double that of unstarved cells (2.7 and 2.1 × 106 cells/mL day, respectively). In contrast to Sstarved cells, N-starved ones were not able to resume growth in the presence of 1 or 2 mg Cr(VI)/L, regardless of the strain, just like unstarved cells (Table 2). However, when subjected to
Table 2 Cell densities (106 cells/mL) of the wild-type and Cr-tolerant strains subjected to a 7-day treatment with 1 and 2 mg Cr(VI)/L after 7 days of culture in standard (unstarved) or in nitrogen deprived (N-starved) medium Cr(VI) (mg/L)
1 2
Wild-type
Cr-tolerant
Unstarved
N-starved
Unstarved
N-starved
3.125 (0.199) 2.945 (0.065)
3.076 (0.034) 2.916 (0.060)
7.281 (0.354) 3.045 (0.094)
9.615 (0.297) 3.089 (0.052)
Means of three replicates. In brackets standard deviation.
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Fig. 3. Dry weight of the wild-type and Cr-tolerant strains: (i) in the stock cultures, (ii) after a 7-day culture in standard (unstarved) or in sulfate-deprived medium (S-starved), (iii) after 24 h recovery from unstarved (S-sufficient) or starved (S-replete) conditions. Error bars = standard deviation; (*) significantly different from unstarved cells (p < 0.01).
7 days of treatment with 1 mg Cr(VI)/L in standard medium, N-starved cells of the tolerant strain reached a significant higher density than the unstarved ones (p < 0.05). 3.3. Dry weight and free Cys, GSH and sulfur content When cultured for 7 days in standard or in S-deprived medium, the dry weight of both strains increased significantly (p < 0.01) (Fig. 3). In S-starved cells it reached values about 3-fold the biomass of stock culture cells. Differences between unstarved and S-starved cells were significant in both strains (p < 0.01). When unstarved and S-starved cells were transferred to and maintained in standard culture medium for 24 h, the dry weight decreased to values close to those of the stock culture cells. Significantly higher dry weight (p < 0.01) than that of unstarved cells was also observed in both strains after 7 days of culture in N-deprived medium (Fig. 4). Differences between the two strains, as well as between S-starved and N-starved cells, were not significant. After 7 days of culture in standard medium, the levels of free Cys and GSH were approximately 0.2 and 1.5 mol SH/g d.w. in the wild-type, and approximately 0.4 and 1.6 mol SH/g d.w. in the Cr-tolerant strain, respectively (Fig. 5). After 7 days of S-
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Fig. 4. Dry weight of the wild-type and Cr-tolerant strains after a 7-day culture in standard (unstarved) or in nitrate-deprived medium (N-starved). Error bars = standard deviation; (*) significantly different from unstarved cells (p < 0.01).
starvation, their levels were approximately 0.08 and 0.45 mol SH/g d.w., respectively, in both strains (Fig. 5). Hence Cys and GSH levels in unstarved and S-starved cells were significantly lower (p < 0.001) than the ones in the stock cultures (0.41 ± 0.06 and 1.95 ± 0.14 mol SH/g d.w. in the wild-type; 1.15 ± 0.13 and 2.61 ± 0.30 mol SH/g d.w. in the Cr-tolerant strain). During the first 24 h of recovery in standard medium, free Cys in S-replete cells significantly increased (p < 0.01) in both strains (Fig. 5). Much of the increase was due to Cys synthesis during the first hour of recovery. Actually, the increment was 0.8 and 1.0 mol SH/g d.w. in S-replete wild-type and Crtolerant cells, respectively. In S-sufficient cells of the Cr-tolerant strain Cys increment, negligible after 1 h (0.25 mol SH/g d.w.), was significant after 24 h recovery (p < 0.05). After 1 and 24 h recovery, Cys levels were significantly higher in S-replete than in S-sufficient cells of both strains (p < 0.05). During the subsequent 24 h recovery, Cys content significantly decreased in S-replete wild-type and in S-sufficient tolerant strain (p < 0.05), while it remained fairly constant in S-sufficient wild-type and in S-replete tolerant strain. However, it must be emphasised that Cys level in the Cr-tolerant strain was still much higher in Sreplete than in S-sufficient cells (p < 0.001) and more than double
Fig. 5. Free cysteine and reduced glutathione content in the wild-type and Cr-tolerant strains just after 7 days of culture in standard or in sulfate-deprived medium (0 h) and after 1, 24 and 48 h recovery from unstarved (S-sufficient) or starved (S-replete) conditions. Error bars = standard deviation; (*) significant difference between the strains.
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Fig. 6. Total sulfur content in the wild-type and Cr-tolerant strains: (i) in the stock cultures, (ii) after a 7-day culture in standard (unstarved) or in sulfate-deprived medium (S-starved), (iii) after 24 h recovery from unstarved (S-sufficient) or starved (S-replete) conditions. Error bars = standard deviation. Different letters label significantly different values (p < 0.01).
the one recorded in the stock culture. After 24 and 48 h recovery, the Cr-tolerant strain showed Cys levels significantly higher than the wild-type (p < 0.05), in both S-sufficient and S-replete cells. GSH content significantly increased (p < 0.01) during the first 24 h of recovery both in S-sufficient and S-replete cells of the two strains (Fig. 5), reaching higher values (p < 0.01) than those of the stock culture. Most of the increment was due to GSH synthesis during the first hour of recovery (1.2 and 1.6 mol SH/g d.w. in S-sufficient and S-replete wild-type, respectively, and 2.0 mol SH/g d.w. in the Cr-tolerant strain). During the subsequent 24 h, GSH content in both S-sufficient and S-replete cells decreased to values close to the ones in the stock cultures. At the various recovery times, differences between S-sufficient and S-replete cells were not significant, with the exception of the tolerant strain after 1 h recovery (p < 0.01). A significant difference between the wild-type and the Cr-tolerant strain (p < 0.05) was recorded in S-sufficient cells after 24 h recovery. Sulfur content in stock culture cells of the wild-type and Crtolerant strains was similar and decreased significantly (p < 0.01) following either S-starvation or a 7-day culture in standard medium (Fig. 6). In particular, after 7 days of starvation, sulfur content was almost half that of stock culture cells. When unstarved and S-starved cells were allowed to recover for 24 h in standard medium, sulfur content increased significantly (p < 0.01) in both strains. In S-sufficient cells, as well as in Sreplete wild type, sulfur reached levels similar to those of the stock cultures, while in S-replete Cr-tolerant strain it reached a mean level which was almost double. Consequently, upon sulfate re-supply after starvation, sulfur in the Cr-tolerant rose to much higher levels (p < 0.001) than in the wild-type strain. 4. Discussion S-starved cells of the two strains of Scenedesmus acutus showed a greater tolerance to Cr(VI) than the unstarved ones.
In particular, the wild-type strain was able to grow in the presence of 3 mg Cr(VI)/L, i.e. three times the concentration which inhibits growth in unstarved cells, and the Cr-tolerant strain maintained the capacity to grow at higher Cr(VI) concentrations than the wild-type. However, the greater the Cr(VI) concentration the smaller the growth of S-starved cells of both strains, thus demonstrating that the increased tolerance was not due to chromium exclusion. This is in agreement with Kleiman and Cogliatti (1997), who observed that chromate uptake increased in S-replete wheat plants, and with Kaszycki et al. (2005), who reported that pre-cultivation at minimum sulfate concentration enhanced the rate of chromium accumulation in Spirodela polyrhiza upon 0.013, 1.0 and 16 mM sulfate re-supply. When S-starved cells were allowed to recover for 3 days in standard medium before Cr(VI) treatment, the wild-type and the Cr-tolerant strain did not grow in the presence of 1 and 2 mg Cr(VI)/L, respectively, suggesting that tolerance after S-starvation was due to physiological transient modifications ascribable to phenotypic plasticity. The increased tolerance to Cr(VI) in the two strains of Scenedesmus acutus after S-starvation and upon sulfate resupply could be ascribed to different causes: (i) an unspecific response to starvation linked to general growth perturbation; (ii) increased synthesis of the thiol compounds (Cys and GSH) involved in counteracting oxidative stress; (iii) increased sulfate uptake and assimilation which are at the basis of Cys and GSH synthesis. Actually, during sulfur or nitrogen starvation, cells of both strains underwent growth perturbation and showed no or almost nil increase in cell density associated with biomass increase. The cells probably remained in a phase that precedes cell division and, when allowed to recover in standard medium, they resumed growth at a high rate with no lag phase. However, following Nstarvation, the sensitivity of the two strains to Cr(VI) did not change. The different effect of S- and N-starvation rules out the hypothesis of a general response to factors perturbing growth and suggests that the transient greater tolerance to Cr(VI) observed after S-starvation is linked to a specific response of the algae to sulfur deprivation. The growth perturbation caused by nitrogen deprivation probably underlies the difference in cell density between unstarved and N-starved cells of the Cr-tolerant strain after a 7-day treatment with 1 mg Cr(VI)/L. In both strains, Cys and GSH content underwent a significant decrease during the 7-day culture in standard medium or in S-deprived medium, but increased rapidly in both S-sufficient and S-replete cells during the first 24 h of recovery in standard medium. Despite the great depletion during starvation, Cys in S-replete cells reached levels which were almost double, and GSH similar to those of S-sufficient cells within the first hour of recovery. Since Cys and GSH are involved in counteracting heavy metal toxicity and oxidative stress, the higher tolerance to Cr(VI) observed after sulfur starvation could be ascribed to this prompt response upon sulfur re-supply. However, it must be stressed that cells were exposed to Cr(VI) just after starvation when the intracellular pools of Cys and GSH and total sulfur were dramatically reduced. Our previous work on the same strains of S. acutus (Gorbi et al., 2006) clearly demonstrated that
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survival to Cr(VI) poisoning depends on the capability to replenish or increase GSH pool, and requires sulfate supply. Looking at the data of sulfur accumulation, it appears that the two strains, after the great depletion suffered during starvation, were able to rapidly restore sulfur levels. The increase of uptake and assimilatory sulfate reduction following S-starvation has been well documented in both algae and higher plants (Yildiz et al., 1994; Smith et al., 1997; Leustek et al., 1994). Yildiz et al. (1994) reported that in normal conditions sulfate uptake in the green alga Chlamidomonas reinhardtii is a typical Michaelis–Menten process which suggests the existence of only one permease. However, the authors observed that both Vmax and K1/2 for sulfate uptake were altered in S-starved cells and suggested that sulfur-deprived C. reinhardtii cells synthesize a new higher affinity sulfate transport system. Studies performed by P´erezCasti˜neira et al. (1998) in the presence of different inhibitors strongly supported multiphasic kinetics for sulfate uptake in C. reinhardtii. In our experiments, both S-starved and unstarved cells of Scenedesmus acutus had to replenish the amount of intracellular sulfur. However, in S-sufficient cells sulfur content increased from ∼0.100 to ∼0.140 g S/106 cells within 24 h of recovery, while in S-replete cells it increased from 0.070 to 0.140 and 0.250 g S/106 in the wild-type and in the Cr-tolerant strain, respectively. It therefore seems that, after starvation, the two strains had a higher capacity for sulfate uptake, i.e. that S-starvation induced an increase of the activity of the mechanisms involved in sulfur uptake and assimilation in both strains. This can be ascribed to the de-repression of the uptake system caused by a lowered intracellular pool of negative metabolic regulators of sulfate transport system. According to Schmidt and Jager (1992) and Smith et al. (1997), sulfate uptake seems to be under a feedback regulation by intracellular sulfate pool or endproducts of sulfate uptake or sulfate assimilation. Herschbach and Rennenberg (1994) and Lappartient and Touraine (1996) suggested that glutathione is involved in the control of SO4 2− uptake in higher plants, the increase of ATP sulfurylase activity and SO4 2− uptake being correlated with a decrease in tissue GSH concentration. Smith et al. (1997) reported that the capacity to take up sulfate increased in barley roots following sulfur starvation and, upon re-supply of sulfate, decreased rapidly concomitantly with the rise in tissue sulfate, Cys and GSH content. Besides, Bolchi et al. (1999) provided evidence “for the glutathione-independent involvement of cysteine as a stereoscopic pretranslational modulator of the expression of sulfur status-responsive genes” in maize. The authors suggested a regulatory model which can allow plants to distinguish between transient variations of GSH levels and a sulfur shortage with compromised protein synthesis. Actually, we observed a drastic reduction in Cys and GSH pools in the two strains of S. acutus when subjected to 7 days of S-starvation, a condition that may have triggered the up-regulation of sulfate uptake/assimilatory process, thus leading to the increased rate of Cys biosynthesis upon sulfate re-supply. The up-regulation of sulfate uptake mechanism following S-starvation can explain the higher tolerance to Cr(VI) displayed by S-starved strains of S. acutus and is consistent with the transient character of the increased tolerance. Since during 24 h of recovery after S-starvation the wild-type
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accumulated a significantly lower amount of sulfur than the Crtolerant strain, it can be hypothesized that the tolerant strain has higher activity/affinity enzymes and/or that the down-regulation of the sulfate uptake/assimilatory process in the wild-type occurs at lower levels of the negative metabolic regulators than in the Crtolerant strain. The latter hypothesis implies that the Cr-tolerant strain is more sensitive to the decrease in the intracellular endproducts of sulfate assimilation processes. It is also consistent with the higher levels of Cys and GSH observed in the stock culture compared to the wild-type strain, as well as the higher level of Cys in S-replete cells. Moreover, it seems to be supported by the rate of increase in free Cys and GSH pools observed in S-replete and S-sufficient cells during the first hour of recovery. Actually, GSH synthesis rate in S-replete cells of the wild-type was almost 50% higher than in S-sufficient cells, while in the Cr-tolerant strain it was in both cases essentially the same and higher than in the wild-type. This suggests that, in the Cr-tolerant strain, even the reduction of GSH pool suffered during 7 days in standard medium is sufficient to induce the increase in GSH synthesis rate to the probably highest value (2.0 mol SH/g d.w. per hour). Considering the levels of both free Cys and GSH, the rate of increase was much higher in S-replete than in S-sufficient cells in both strains and in the Cr-tolerant strain than in the wild type. This different behaviour can explain the differences in tolerance between the two strains despite their similar GSH content just after starvation and during 48 h recovery. These findings in the unicellular alga S. acutus seem to be in agreement with the regulatory model suggested by Bolchi et al. (1999) for higher plants. Higher sensitivity to the decrease in the levels of intracellular end-products of sulfate assimilation processes leading to an earlier up-regulation could thus be regarded as a primary reason of the tolerance to chromium in the selected strain of S. acutus.
5. Conclusions Sulfur-starved cells of the wild-type and Cr-tolerant strains of Scenedesmus acutus showed a higher tolerance to Cr(VI) than unstarved cells. The increased tolerance observed upon sulfate re-supply was due to physiological transient modifications ascribable to phenotypic plasticity and can be regarded as a consequence of specific responses of the algae to sulfur deprivation. Actually, after starvation, the two strains had a higher capacity for sulfur uptake and were able to rapidly restore GSH pool and increase free Cys to levels almost twice those of unstarved cells. These responses suggest that the higher tolerance to Cr(VI) after S-starvation is linked to the up-regulation of the sulfate uptake/assimilatory process. The greater sulfur uptake and rate of increase in free Cys and GSH content after starvation suggest that the Cr-tolerant strain may have a higher sensitivity to the decrease in the levels of intracellular end-products of sulfate assimilation processes than the wild-type. This can lead to an earlier up-regulation of these processes and could constitute a primary reason of the tolerance to chromium in the selected strain of S. acutus.
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Acknowledgements This work was supported by University of Parma grant (FIL 2003–2004). References Bolchi, A., Petrucco, S., Tenca, P.L., Foroni, C., Ottonello, S., 1999. Coordinate modulation of maize sulfate and ATP sulfurylase mRNAs in response to variations in sulfur nutritional status: stereospecific down-regulation by lcysteine. Plant Mol. Biol. 39, 527–537. Clarkson, D.T., Saker, L.R., 1989. Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient. Planta 178, 249–257. Corradi, M.G., Gorbi, G., Bassi, M., 1995a. Hexavalent chromium induces gametogenesis in the freshwater alga Scenedesmus acutus. Ecotoxicol. Environ. Saf. 30, 106–110. Corradi, M.G., Gorbi, G., Ricci, A., Torelli, A., Bassi, M., 1995b. Chromiuminduced sexual reproduction gives rise to a Cr-tolerant progeny in Scenedesmus acutus. Ecotoxicol. Environ. Saf. 32, 12–18. Corradi, M.G., Gorbi, G., Morsi Abd-El-Monem, H., Torelli, A., Bassi, M., 1998. Exudates from the wild-type and a Cr-tolerant strain of Scenedesmus acutus influence differently Cr(VI) toxicity to algae. Chemosphere 37, 3019–3025. Deane-Drummond, C.E., 1987. The regulation of sulphate uptake following growth of Pisum sativum L. seedlings in S nutrient limiting conditions. Interaction between nitrate and sulphate transport. Plant Sci. 50, 27–35. Gaur, J.P., Rai, L.C., 2001. Heavy metal tolerance in algae. Algal adaptation to environmental stresses. In: Rai, L.C., Gaur, J. (Eds.), Physiological, Biochemical and Molecular Mechanisms. P. Springer-Verlag Berlin Heidelberg, New York, pp. 363–388. Gekeler, W., Grill, E., Winnacker, E.-L., Zenk, M.H., 1988. Algae sequester heavy metals via synthesis of phytochelatin complexes. Arch. Microbiol. 150, 197–202. Gorbi, G., Corradi, M.G., Invidia, M., Rivara, L., Bassi, M., 2002. Is Cr(VI) toxicity to Daphnia magna modified by food availability or algal exudates? The hypothesis of a specific chromium/algae/exudates interaction. Water Res. 36, 1917–1926. Gorbi, G., Torricelli, E., Pawlik-Skowro´nska, B., Sanit`a di Toppi, L., Zanni, C., Corradi, M.G., 2006. Differential responses to Cr(VI)-induced oxidative stress between Cr-tolerant and wild-type strains of Scenedesmus acutus (Chlorophyceae). Aquat. Toxicol. 79, 132–139. Herschbach, C., Rennenberg, H., 1994. Influence of glutathione (GSH) on net uptake of sulfate and sulfate transport in tobacco plants. J. Exp. Bot. 45, 1069–1076. Hu, S., Lau, K.W.K., Wu, M., 2001. Cadmium sequestration in Chlamydomonas reinhardtii. Plant Sci. 161, 987–996. Kaszycki, P., Gabry´s, H., Appenroth, K.-J., Jaglarz, A., Sedziwy, S., Walczack, T., Koloczek, H., 2005. Exogenously applied sulphate as a tool to investigate transport and reduction of chromate in the duckweed Spirodela polyrhiza. Plant Cell Environ. 28, 260–268. Kleiman, I.D., Cogliatti, D.H., 1997. Uptake of chromate in sulfate deprived wheat plants. Environ. Pollut. 97, 131–135. Lappartient, A.G., Touraine, B., 1996. Demand-driven control of root ATP sulfurylase activity and SO4 2− uptake in intact canola. Plant Physiol. 111, 147–157. Leustek, T., Murillo, M., Cervantes, M., 1994. Cloning of a cDNA encoding ATP sulfurylase Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae. Plant Physiol. 105, 897–902. Nocito, F.F., Pirovano, L., Cocucci, M., Sacchi, G.A., 2002. Cadmium-induced sulphate uptake in maize roots. Plant Physiol. 129, 1872–1879.
Pankow, J.F., Janauer, G.E., 1974. Analysis for chromium traces in natural water. Part I. Preconcentration of chromate from p.p.b. levels in aqueous solutions by ion exchange. Anal. Chim. Acta 69, 97–104. Pawlik-Skowro´nska, B., Skowro´nski, T., 2001. Freshwater algae. In: Prasad, M.N.V. (Ed.), Metals in the Environment. Marcel Dekker, New York, pp. 59–94. Pawlik-Skowro´nska, B., 2003a. Resistance, accumulation and allocation of zinc in two ecotypes of the green alga Stigeoclonium tenue K¨utz. coming from habitats of different heavy metal concentrations. Aquat. Bot. 75, 189– 198. Pawlik-Skowro´nska, B., 2003b. When adapted to high zinc concentrations the periphytic green alga Stigeoclonium tenue produces high amounts of novel phytochelatin-related peptides. Aquat. Toxicol. 62, 155–163. P´erez-Casti˜neira, J.R., Prieto, J.L., Gonz´alez-Arroyo, J.G., Vega, J.M., 1998. Kinetic properties of sulfate uptake in two types of eukaryotic green microalgae. J. Plant Physiol. 153, 324–331. Perez-Rama, M., Herrero Lopez, C., Abalde Alonso, J., Torres Vamonde, E., 2001. Class III metallothioneins in response to cadmium toxicity in the marine microalga Tetraselmis suecica (Kylin) Butch. Environ. Toxicol. Chem. 20, 2061–2066. Rauser, W.E., 1999. Structure and function of metal chelators produced by plants. Cell Biochem. Biophys. 31, 19–49. Raven, J.A., 1980. Nutrient transport in microalgae. Adv. Microb. Physiol. 20, 47–226. Reese, R.N., Mehra, R.K., Tarbet, E.B., Winge, D.R., 1988. Studies on the ␥-glutamyl Cu-binding peptide from Schizosaccharomyces pombe. J. Biol. Chem. 263, 4186–4192. Reese, R.N., Winge, D.R., 1988. Sulfide stabilization of the cadmium-␥glutamyl peptide complex of Schizosaccharomyces pombe. J. Biol. Chem. 263, 12832–12835. Riedel, G.F., 1985. The relationship between chromium(VI) uptake, sulfate uptake and chromium(VI) toxicity in the estuarine diatom Thalassiosira pseudonana. Aquat. Toxicol. 7, 191–204. Sanit`a di Toppi, L., Gremigni, P., Pawlik-Skowro´nska, B., Prasad, M.N.V., Cobbett, C.S., 2003. Response to heavy metals in plants: a molecular approach. In: Sanit`a di Toppi, L., Pawlik-Skowro´nska, B. (Eds.), Abiotic Stresses in Plants. Kluwer Academic Publishers, Dordrecht, pp. 133–156. Schmidt, A., Jager, K., 1992. Open question about sulfur metabolism in plants. Annu. Rev. Plant Physiol. 43, 325–349. Skeffington, R.A., Shewry, P.R., Peterson, P.J., 1976. Chromium uptake and transport in barley seedlings (Hordeum vulgare L.). Planta 132, 209– 214. Smith, F.W., Hawkesford, M.J., Ealing, P.M., Clarkson, D.T., Vanden Berg, P.J., Belcher, A.R., Warrilow, A.G.S., 1997. Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter. Plant J. 12, 875–884. Speiser, D.M., Abrahamson, S.L., 1992. Brassica juncea produces a phytochelatin-cadmium-sulfide complex. Plant Physiol. 99, 817–821. Torres, E., Cid, A., Fidalgo, P., Herrero, C., Abalde, J., 1997. Long-chain class III metallothioneins as a mechanisms of cadmium tolerance in the marine diatom Phaeodactilum tricornutum Bohlin. Aquat. Toxicol. 39, 231–246. Torricelli, E., Gorbi, G., Pawlik-Skowronska, B., Sanit`a di Toppi, L., Corradi, M.G., 2004. Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus. Aquat. Toxicol. 68, 315–323. US Environmental Protection Agency (EPA), 1971. Algal assay procedure bottle test. National Eutrophication Research Program. Pacific Northwest Environmental Research Laboratory, Corvallis, OR. Yildiz, F.H., Davies, J.P., Grossman, A.R., 1994. Characterization of sulfate transport in Chlamydomonas reinhardtii during sulfur-limited and sulfursufficient growth. Plant Physiol. 104, 981–987.
Sulfur starvation and chromium tolerance in Scenedesmus acutus: A possible link between metal tolerance and the regulation of sulfur uptake/assimilation processes Gessica Gorbi a , Corrado Zanni b , Maria Grazia Corradi b,∗ a
b
Department of Environmental Sciences, University of Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy Department of Evolutionary and Functional Biology, University of Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy Received 2 May 2007; received in revised form 11 July 2007; accepted 17 July 2007
Abstract In a laboratory-selected Cr-tolerant strain of the unicellular green alga Scenedesmus acutus, the capacity to synthesize higher amounts of cysteine (Cys) and reduced glutathione (GSH) than the wild-type was demonstrated to underlie tolerance to Cd and Cr(VI). In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys, hence it was hypothesized that the sensitivity of the two strains to Cr(VI) could be modified after culturing in sulfate-deprived medium. Both strains were grown in the presence of different concentrations or in the absence of sulfate (sulfur-starved) and then assayed for Cr(VI) tolerance in standard medium. Unstarved, sulfur-starved and sulfur-replete cells (cells maintained in standard medium after S-starvation) were analysed for Cys, GSH and sulfur content. Sulfur-starved cells showed a greater tolerance to Cr(VI) than unstarved ones. The increased tolerance was ascribable to a transient physiological change and can be considered as specifically due to sulfur deprivation, since it was lost after a 3-day culture in standard medium and was not exhibited by nitrogen-starved cells. The comparison between Cys, GSH and sulfur content in sulfur-starved and sulfur-replete cells of the two strains suggests that the higher tolerance to Cr(VI) after S-starvation could depend on the up-regulation of sulfate uptake mechanisms, and that the primary reason for the higher tolerance to chromium in the selected strain could be due to greater sensitivity to the decrease in negative intracellular end-products (free Cys and GSH) leading to an earlier up-regulation of sulfate assimilation processes. © 2007 Elsevier B.V. All rights reserved. Keywords: Cr tolerance; Sulfur starvation; Free cysteine; Glutathione; Sulfur content; Scenedesmus acutus
1. Introduction Among the mechanisms involved in heavy metal detoxification in algae, synthesis of reduced glutathione (GSH) and phytochelatins (PCs) plays an important role (Gekeler et al., 1988; Gaur and Rai, 2001; Pawlik-Skowro´nska and Skowro´nski, 2001; Sanit`a di Toppi et al., 2003). Although various studies have focused on this topic, the role of thiol peptides in tolerance to heavy metals in algae is not fully recognised. Torres et al. (1997) ascribed the high tolerance of Cd in Phaeodactylum tricornutum to an increased production of PCs having higher molecular weight compared to other algal species. In addition, Perez-Rama et al. (2001) suggested that the high tolerance to Cd in Tetraselmis suecica could be due to its ability
∗
Corresponding author. Tel.: +39 0521 905597; fax: +39 0521 905403. E-mail address: [email protected] (M.G. Corradi).
0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2007.07.006
to synthesize longer PCs than other algal species. Hu et al. (2001) observed that a Cd-resistant strain of Chlamydomonas reinhardtii produced and maintained significantly higher levels of free Cys than the sensitive strain. The authors suggested that Cys synthesis/turnover and production of high molecular weight (HMW) PC–metal complexes can be linked. Rauser (1999) reported that HMW-complexes contain acid labile sulfide (S2− ) which gives them increased stability and higher metalbinding capacity, as suggested also by Reese et al. (1988) and Reese and Winge (1988). Speiser and Abrahamson (1992) proposed Cys sulfinate as a source of sulfide to be incorporated in PC–metal HMW complex. Hence, besides being the structural thiol aminoacid of GSH and PCs, Cys seems to have an additional role in tolerance to Cd. According to Pawlik-Skowro´nska (2003a,b) the tolerance to high concentrations of zinc in a Zn-tolerant ecotype of the filamentous green alga Stigeoclonium tenue relies on the complexation of metal with Cys-rich derivatives of phytochelatins, novel phytochelatin-related com-
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pounds produced in larger amounts than in the sensitive ecotype. In previous studies, two strains of the unicellular green alga Scenedesmus acutus, namely a wild-type and a laboratoryselected strain tolerant to Cr(VI), Cu and Cd, were taken as a model for the analysis of the mechanisms involved in metal detoxification and tolerance in algae (Corradi et al., 1995a,b, 1998; Gorbi et al., 2002; Torricelli et al., 2004). The tolerant strain showed higher constitutive levels of free Cys than the wildtype strain and significantly higher levels of Cys, GSH and PCs after treatment with Cd (Torricelli et al., 2004). In the tolerant strain, Cys content increased with the increase of Cd concentration and reached a level 10-fold higher than the constitutive one. These results suggested that the ability of the tolerant strain to synthesize high amount of Cys may be a key mechanism of defence against Cd toxicity. Further studies (Gorbi et al., 2006) demonstrated that when subjected to Cr(VI) treatment, the tolerant strain is able to maintain higher GSH levels than the wild type. The comparison of the recovery capacity and GSH levels in the two strains after Cr treatment in sulfate-enriched and -deprived condition clearly demonstrated that sulfate availability influences survival and suggested that tolerance to Cr(VI) is related to GSH biosynthetic capacity. In photosynthetic organisms, sulfate constitutes the main source of sulfur for the biosynthesis of cysteine. In green algae, Cys synthase is regulated; however, Cys increase can be detected only after prolonged sulfur starvation probably because it can be supplied by degradation of glutathione (Schmidt and Jager, 1992). Algae possess an active transport system for sulfate, whose uptake increases after prolonged sulfur starvation (Raven, 1980). Pankow and Janauer (1974) suggested that Cr(VI) toxicity might be due to chromate (CrO4 2− ) anion interfering with the uptake of sulfate (SO4 2− ) anion, which has similar size. Skeffington et al. (1976) reported that sulfate and chromate uptake appears to rely on the same transport system in Hordeum vulgare. Riedel (1985) observed that Cr(VI) uptake in Thalassiosira pseudonana was inversely proportional to external sulfate and Cr(VI) concentrations inhibiting growth also inhibited sulfate uptake. According to Riedel (1985), the result of the competition between sulfate and chromate for uptake is that increasing sulfate concentration promotes increased tolerance and decreased uptake of hexavalent chromium for phytoplankton at least. Deane-Drummond (1987) and Clarkson and Saker (1989) observed that sulfate uptake increased in sulfate-deprived plants and a new or modified sulfate transport system having a higher affinity for sulfate was induced. Yildiz et al. (1994) characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfursufficient and sulfur-deficient conditions and observed that sulfur-deprived cells synthesized a new, high-affinity sulfate transport system which accumulated rapidly and was detectable in cells within 1 h of sulfur deprivation. Kleiman and Cogliatti (1997) reported that when the sulfate transport system is derepressed by a previous sulfate-deprivation, a higher chromate uptake in the presence of sulfate can be observed in Triticum aestivum exposed to high Cr(VI) concentrations. Besides, sulfate uptake in Chlamydomonas reinhardtii and Monoraphidium
braunii is inhibited by anions structurally similar to sulfate such as chromate, selenate and molybdate (P´erez-Casti˜neira et al., 1998). Nocito et al. (2002) observed a 100% increase in sulfate uptake, associated with a decrease in sulfate and GSH content and synthesis of large amounts of PCs in Cd-treated maize roots. These studies indicate that the amount of sulfur available can be a factor influencing the response of algae to chromium. The aim of the present study is to evaluate if the sensitivity of the wild-type and the Cr-tolerant strains of Scenedesmus acutus to hexavalent chromium is modified by culturing in sulfur-limited or sulfur-deprived conditions and to identify which physiological and/or biochemical alterations can be responsible for the differences in sensitivity. The two strains were grown in the presence of different concentrations or in the absence of sulfate, and then treated with Cr(VI) in standard medium. Variations in Cys, GSH and sulfur content in response to sulfur deprivation and repletion were analysed. To evaluate if the response of sulfurstarved algae to chromium can be considered as specifically due to sulfur deprivation, the two strains were also subjected to Cr(VI) treatment after culturing in nitrogen-deprived medium. The primary reason of Cr tolerance in the selected strain is discussed. 2. Materials and methods Culturing of Scenedesmus acutus strains and experiments were carried out in a climate-controlled chamber, at 24 ± 1 ◦ C, 230 mol/m2 s and 16-h photoperiod. Population growth during treatments or recovery was checked by cell counting with a Neubauer haemocytometer by light microscopy. 2.1. Sulfur starvation and Cr tolerance Synchronized axenic stock cultures (initial cell density 3 × 106 cells/mL) of the two strains of Scenedesmus acutus, i.e. the wild-type and the Cr-tolerant strains, were grown for 3 days in 2 L Erlenmeyer flasks in EPA (1971) liquid culture medium modified by lowering zinc concentration to 3.14 g/L. Taking into account the high cell density, salt concentrations were doubled. Aliquots of the stock cultures, in exponential growth phase, were centrifuged for 10 min at 2200 × g and washed with distilled water. The pellets were suspended at 3 × 106 cells/mL density in the standard culture medium (200 mL) or in the medium containing 25, 3 or 0% of the standard sulfate amount (14.36 mg MgSO4 /L). Since MgSO4 is the only source of sulfur in the standard medium, the amount of MgCl2 was simultaneously increased to restore standard magnesium concentration. After 1-, 2-, 4- and 7-day culture, cell density was assessed. After 7-day culture under the different sulfate concentrations, cells of both strains were collected by centrifugation, washed and treated with the lowest Cr(VI) concentration inhibiting growth, i.e. 1 mg/L for the wild-type and 2 mg/L for the Cr-tolerant strain. Treatments with chromium, supplied as K2 Cr2 O7 , were carried out in standard medium (200 mL) at initial cell density of 3 × 106 cells/mL and, after 1, 2, 4 and 7 days of treatment, cell density was checked. The cells cultured in sulfate-deprived medium (0%
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S) were treated with 2, 3, 4 or 5 mg Cr(VI)/L as described above. Also, after 7 days of culture in standard medium (unstarved) or in sulfate-deprived medium (S-starved), the cells of both strains were centrifuged, re-suspended in standard fresh medium at 3 × 106 cells/mL density and cultured for 3 days. Afterwards the cells were treated with 1 or 2 mg Cr(VI)/L, depending on the strain, as described above. 2.2. Nitrogen starvation and Cr tolerance Aliquots of the stock cultures of the two strains, in exponential growth phase, were centrifuged for 10 min at 2200 × g and washed with distilled water. The pellets were suspended in standard or in nitrogen-deprived medium to obtain an initial density of 3 × 106 cells/mL and cell density was assessed after 1, 2, 4 and 7 days. Since nitrogen was supplied as NaNO3 , sodium concentration in N-deprived medium was restored by increasing the amount of NaHCO3 appropriately. After the 7-day culture, both strains were treated with 1 and 2 mg Cr(VI)/L in standard medium as described above. 2.3. Dry weight and free cysteine (Cys), reduced glutathione (GSH) and sulfur content Unstarved and S-starved cells of both strains were collected by centrifugation, washed, re-suspended in standard medium at 3 × 106 cells/mL density and cultured for 1, 24 and 48 h at the same conditions as above. From now on, the term “S-replete cells” will be used to indicate S-starved cells which were allowed to recover in standard medium for 1, 24 and 48 h, while the term “S-sufficient cells” will be used to indicate unstarved cells transferred to and maintained in standard medium. Dry weight and free Cys and GSH levels were determined in unstarved, Sstarved, S-replete and S-sufficient cells after 1, 24 and 48 h of recovery, as well as in the cells of the stock cultures. Sulfur content was also determined after 24 h of recovery. The dry weight of the two strains after 7-day culture in N-deprived medium was checked too. For dry weight determination, aliquots (90 mL) of the cultures were filtered on pre-weighed mixed cellulose ester filters with pore size of 0.45 m. The filters were washed three times with doubly distilled water, dried at 95 ◦ C for at least 3 h and weighed. Other aliquots of the same cultures (10 mL) were used to determine cell density by cell counting. Dry weight of 106 cells was calculated. For free Cys and GSH determination, cells were harvested by filtration on a Whatman GF/C filter and extracted using a mortar in ice-cold 5% (w/v) 5-sulfosalicylic acid solution (SSA) with 6.3 mM diethylenetriaminopentaacetic acid (DTPA). After centrifugation at 14,000 × g for 30 min, total non-protein thiol compounds were determined spectrophotometrically at 412 nm in a mixture of 300 L of the supernatants, 1200 L phosphate buffer (pH 7.6) and 25 L Ellman’s reagent. For free Cys determination, 100 L of phosphate buffer were replaced by 0.1 M methyl glyoxal which binds Cys and, after a 10-min reaction, absorbance was determined at the same wave length as above. Cysteine concentration was calculated as the difference between
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total SH and GSH concentration. A calibration curve for standard SH groups (l-Cysteine, MERCK) was used for quantitative determination of Cys and GSH in the extracts. The efficiency of methyl glyoxal in masking Cys was checked on mixtures of Cys and GSH in different proportions. For sulfur determination, 1500 mL of each culture were centrifuged and washed three times with distilled water. The pellet was dried at 95 ◦ C and then turned to powder in a mortar. Subsamples of 15 mg were weighed in tin cups and, after the addition of 10 mg of vanadium pentoxide (V2 O5 ), subjected to controlled combustion at 900 ◦ C. Total sulfur in the combustion products was determined by gas chromatography (carrier gas = He). 2.4. Statistical analysis All experiments were repeated at least three times with the only exception of growth evaluation under different sulfate concentrations. The significance of the differences in growth between the wild-type and the Cr-tolerant strain subjected to Cr treatments after S-starvation was tested by one-way ANOVA after verification of variance homogeneity (Levene’ test). To evaluate the significance of the differences among treatments and between the two strains subjected to the same treatment, dry weight, total sulfur, Cys and GSH data were subjected to one-way ANOVA after verification of variance homogeneity by Levene’ test. Multiple comparison Tukey test was applied when appropriate. 3. Results 3.1. Sulfur starvation and Cr tolerance In the presence of 25% sulfate, the growth of the two strains of Scenedesmus acutus was the same as controls, while it was almost completely inhibited at lower concentration (3%) or in the absence of sulfate (0% S) (Fig. 1). However, after the 7day culture in S-limited (3% S) or -deprived (0% S) medium, the wild-type and the Cr-tolerant strains resumed growth when transferred to standard culture medium (data not shown) and were able to grow even in the presence of Cr(VI) concentrations (i.e. 1 and 2 mg/L, respectively) which inhibited growth of unstarved (100% S) and 25% S cells (Table 1). In addition, after culturing for 7 days in S-deprived medium (S-starvation), the wild-type strain was also able to grow in the presence of 3 mg Cr(VI)/L, reaching a density of approx 5 × 106 cells/mL after a lag phase of 1 day, and the Cr-tolerant strain was not completely inhibited at 4 mg Cr(VI)/L (Fig. 2). Even if able to grow in the presence of Cr(VI) concentration that inhibited unstarved cells, the greater the Cr(VI) concentration, the weaker the growth of S-starved cells (Fig. 2). However, the Cr-tolerant strain reached significantly higher cell densities (p < 0.01) than the wild-type at all Cr(VI) concentrations tested, except for the highest. When allowed to recover for 3 days in standard medium after S-starvation (3 days S-replete), cells of both strains lost the acquired tolerance, undergoing complete growth inhibition in the presence of 1 or 2 mg Cr(VI)/L (Table 1).
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Fig. 1. Growth of the wild-type and Cr-tolerant strains of Scenedesmus acutus in the presence of different concentrations of sulfate (S) expressed as percentage of the standard medium concentration (100% S). Data from a single experiment. Table 1 Cell densities (106 cells/mL) of the wild-type and Cr-tolerant strains after a 7-day treatment with Cr(VI) Strain
Wild-type (1 mg Cr(VI)/L) Cr-tolerant (2 mg Cr(VI)/L)
Pre-cultivation 100% S
25% S
3% S
0% S
3 days S-replete
3.13 (0.20) 3.05 (0.09)
3.06 (0.04) 3.16 (0.04)
8.99 (0.07) 7.97 (0.27)
8.64 (0.44) 9.59 (0.46)
3.07 (0.10) 3.19 (0.04)
Before Cr-treatment, cells of both strains were pre-cultivated as follows: (i) 7 days in standard growth medium (100% S) or in the medium containing 25%, 3% or 0% sulfate (S), (ii) 7 days in S-deprived medium followed by a 3-day recovery in standard medium (3 days S-replete). After pre-cultivation, the cells were collected, suspended in standard medium (3 × 106 cells/mL) supplemented with 1 or 2 mg Cr(VI)/L, depending on the strain. Means of three replicates. In brackets standard deviation.
Fig. 2. Growth of the wild-type and Cr-tolerant strains of Scenedesmus acutus in the presence of various Cr(VI) concentrations after 7 days of pre-cultivation in sulfate-deprived medium. After pre-cultivation the cells were collected, suspended in standard growth medium (3 × 106 cells/mL) and supplemented with different Cr(VI) concentrations. Error bars = standard deviation.
3.2. Nitrogen starvation and Cr tolerance Culturing the cells in the absence of nitrogen completely inhibited cell multiplication in both strains. Following transfer to standard nitrogen conditions, N-starved cells of the wild-type and Cr-tolerant strains started growing immediately and, dur-
ing the first two days, showed a growth rate (4.2 and 3.7 × 106 cells/mL day, respectively) almost double that of unstarved cells (2.7 and 2.1 × 106 cells/mL day, respectively). In contrast to Sstarved cells, N-starved ones were not able to resume growth in the presence of 1 or 2 mg Cr(VI)/L, regardless of the strain, just like unstarved cells (Table 2). However, when subjected to
Table 2 Cell densities (106 cells/mL) of the wild-type and Cr-tolerant strains subjected to a 7-day treatment with 1 and 2 mg Cr(VI)/L after 7 days of culture in standard (unstarved) or in nitrogen deprived (N-starved) medium Cr(VI) (mg/L)
1 2
Wild-type
Cr-tolerant
Unstarved
N-starved
Unstarved
N-starved
3.125 (0.199) 2.945 (0.065)
3.076 (0.034) 2.916 (0.060)
7.281 (0.354) 3.045 (0.094)
9.615 (0.297) 3.089 (0.052)
Means of three replicates. In brackets standard deviation.
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Fig. 3. Dry weight of the wild-type and Cr-tolerant strains: (i) in the stock cultures, (ii) after a 7-day culture in standard (unstarved) or in sulfate-deprived medium (S-starved), (iii) after 24 h recovery from unstarved (S-sufficient) or starved (S-replete) conditions. Error bars = standard deviation; (*) significantly different from unstarved cells (p < 0.01).
7 days of treatment with 1 mg Cr(VI)/L in standard medium, N-starved cells of the tolerant strain reached a significant higher density than the unstarved ones (p < 0.05). 3.3. Dry weight and free Cys, GSH and sulfur content When cultured for 7 days in standard or in S-deprived medium, the dry weight of both strains increased significantly (p < 0.01) (Fig. 3). In S-starved cells it reached values about 3-fold the biomass of stock culture cells. Differences between unstarved and S-starved cells were significant in both strains (p < 0.01). When unstarved and S-starved cells were transferred to and maintained in standard culture medium for 24 h, the dry weight decreased to values close to those of the stock culture cells. Significantly higher dry weight (p < 0.01) than that of unstarved cells was also observed in both strains after 7 days of culture in N-deprived medium (Fig. 4). Differences between the two strains, as well as between S-starved and N-starved cells, were not significant. After 7 days of culture in standard medium, the levels of free Cys and GSH were approximately 0.2 and 1.5 mol SH/g d.w. in the wild-type, and approximately 0.4 and 1.6 mol SH/g d.w. in the Cr-tolerant strain, respectively (Fig. 5). After 7 days of S-
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Fig. 4. Dry weight of the wild-type and Cr-tolerant strains after a 7-day culture in standard (unstarved) or in nitrate-deprived medium (N-starved). Error bars = standard deviation; (*) significantly different from unstarved cells (p < 0.01).
starvation, their levels were approximately 0.08 and 0.45 mol SH/g d.w., respectively, in both strains (Fig. 5). Hence Cys and GSH levels in unstarved and S-starved cells were significantly lower (p < 0.001) than the ones in the stock cultures (0.41 ± 0.06 and 1.95 ± 0.14 mol SH/g d.w. in the wild-type; 1.15 ± 0.13 and 2.61 ± 0.30 mol SH/g d.w. in the Cr-tolerant strain). During the first 24 h of recovery in standard medium, free Cys in S-replete cells significantly increased (p < 0.01) in both strains (Fig. 5). Much of the increase was due to Cys synthesis during the first hour of recovery. Actually, the increment was 0.8 and 1.0 mol SH/g d.w. in S-replete wild-type and Crtolerant cells, respectively. In S-sufficient cells of the Cr-tolerant strain Cys increment, negligible after 1 h (0.25 mol SH/g d.w.), was significant after 24 h recovery (p < 0.05). After 1 and 24 h recovery, Cys levels were significantly higher in S-replete than in S-sufficient cells of both strains (p < 0.05). During the subsequent 24 h recovery, Cys content significantly decreased in S-replete wild-type and in S-sufficient tolerant strain (p < 0.05), while it remained fairly constant in S-sufficient wild-type and in S-replete tolerant strain. However, it must be emphasised that Cys level in the Cr-tolerant strain was still much higher in Sreplete than in S-sufficient cells (p < 0.001) and more than double
Fig. 5. Free cysteine and reduced glutathione content in the wild-type and Cr-tolerant strains just after 7 days of culture in standard or in sulfate-deprived medium (0 h) and after 1, 24 and 48 h recovery from unstarved (S-sufficient) or starved (S-replete) conditions. Error bars = standard deviation; (*) significant difference between the strains.
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Fig. 6. Total sulfur content in the wild-type and Cr-tolerant strains: (i) in the stock cultures, (ii) after a 7-day culture in standard (unstarved) or in sulfate-deprived medium (S-starved), (iii) after 24 h recovery from unstarved (S-sufficient) or starved (S-replete) conditions. Error bars = standard deviation. Different letters label significantly different values (p < 0.01).
the one recorded in the stock culture. After 24 and 48 h recovery, the Cr-tolerant strain showed Cys levels significantly higher than the wild-type (p < 0.05), in both S-sufficient and S-replete cells. GSH content significantly increased (p < 0.01) during the first 24 h of recovery both in S-sufficient and S-replete cells of the two strains (Fig. 5), reaching higher values (p < 0.01) than those of the stock culture. Most of the increment was due to GSH synthesis during the first hour of recovery (1.2 and 1.6 mol SH/g d.w. in S-sufficient and S-replete wild-type, respectively, and 2.0 mol SH/g d.w. in the Cr-tolerant strain). During the subsequent 24 h, GSH content in both S-sufficient and S-replete cells decreased to values close to the ones in the stock cultures. At the various recovery times, differences between S-sufficient and S-replete cells were not significant, with the exception of the tolerant strain after 1 h recovery (p < 0.01). A significant difference between the wild-type and the Cr-tolerant strain (p < 0.05) was recorded in S-sufficient cells after 24 h recovery. Sulfur content in stock culture cells of the wild-type and Crtolerant strains was similar and decreased significantly (p < 0.01) following either S-starvation or a 7-day culture in standard medium (Fig. 6). In particular, after 7 days of starvation, sulfur content was almost half that of stock culture cells. When unstarved and S-starved cells were allowed to recover for 24 h in standard medium, sulfur content increased significantly (p < 0.01) in both strains. In S-sufficient cells, as well as in Sreplete wild type, sulfur reached levels similar to those of the stock cultures, while in S-replete Cr-tolerant strain it reached a mean level which was almost double. Consequently, upon sulfate re-supply after starvation, sulfur in the Cr-tolerant rose to much higher levels (p < 0.001) than in the wild-type strain. 4. Discussion S-starved cells of the two strains of Scenedesmus acutus showed a greater tolerance to Cr(VI) than the unstarved ones.
In particular, the wild-type strain was able to grow in the presence of 3 mg Cr(VI)/L, i.e. three times the concentration which inhibits growth in unstarved cells, and the Cr-tolerant strain maintained the capacity to grow at higher Cr(VI) concentrations than the wild-type. However, the greater the Cr(VI) concentration the smaller the growth of S-starved cells of both strains, thus demonstrating that the increased tolerance was not due to chromium exclusion. This is in agreement with Kleiman and Cogliatti (1997), who observed that chromate uptake increased in S-replete wheat plants, and with Kaszycki et al. (2005), who reported that pre-cultivation at minimum sulfate concentration enhanced the rate of chromium accumulation in Spirodela polyrhiza upon 0.013, 1.0 and 16 mM sulfate re-supply. When S-starved cells were allowed to recover for 3 days in standard medium before Cr(VI) treatment, the wild-type and the Cr-tolerant strain did not grow in the presence of 1 and 2 mg Cr(VI)/L, respectively, suggesting that tolerance after S-starvation was due to physiological transient modifications ascribable to phenotypic plasticity. The increased tolerance to Cr(VI) in the two strains of Scenedesmus acutus after S-starvation and upon sulfate resupply could be ascribed to different causes: (i) an unspecific response to starvation linked to general growth perturbation; (ii) increased synthesis of the thiol compounds (Cys and GSH) involved in counteracting oxidative stress; (iii) increased sulfate uptake and assimilation which are at the basis of Cys and GSH synthesis. Actually, during sulfur or nitrogen starvation, cells of both strains underwent growth perturbation and showed no or almost nil increase in cell density associated with biomass increase. The cells probably remained in a phase that precedes cell division and, when allowed to recover in standard medium, they resumed growth at a high rate with no lag phase. However, following Nstarvation, the sensitivity of the two strains to Cr(VI) did not change. The different effect of S- and N-starvation rules out the hypothesis of a general response to factors perturbing growth and suggests that the transient greater tolerance to Cr(VI) observed after S-starvation is linked to a specific response of the algae to sulfur deprivation. The growth perturbation caused by nitrogen deprivation probably underlies the difference in cell density between unstarved and N-starved cells of the Cr-tolerant strain after a 7-day treatment with 1 mg Cr(VI)/L. In both strains, Cys and GSH content underwent a significant decrease during the 7-day culture in standard medium or in S-deprived medium, but increased rapidly in both S-sufficient and S-replete cells during the first 24 h of recovery in standard medium. Despite the great depletion during starvation, Cys in S-replete cells reached levels which were almost double, and GSH similar to those of S-sufficient cells within the first hour of recovery. Since Cys and GSH are involved in counteracting heavy metal toxicity and oxidative stress, the higher tolerance to Cr(VI) observed after sulfur starvation could be ascribed to this prompt response upon sulfur re-supply. However, it must be stressed that cells were exposed to Cr(VI) just after starvation when the intracellular pools of Cys and GSH and total sulfur were dramatically reduced. Our previous work on the same strains of S. acutus (Gorbi et al., 2006) clearly demonstrated that
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survival to Cr(VI) poisoning depends on the capability to replenish or increase GSH pool, and requires sulfate supply. Looking at the data of sulfur accumulation, it appears that the two strains, after the great depletion suffered during starvation, were able to rapidly restore sulfur levels. The increase of uptake and assimilatory sulfate reduction following S-starvation has been well documented in both algae and higher plants (Yildiz et al., 1994; Smith et al., 1997; Leustek et al., 1994). Yildiz et al. (1994) reported that in normal conditions sulfate uptake in the green alga Chlamidomonas reinhardtii is a typical Michaelis–Menten process which suggests the existence of only one permease. However, the authors observed that both Vmax and K1/2 for sulfate uptake were altered in S-starved cells and suggested that sulfur-deprived C. reinhardtii cells synthesize a new higher affinity sulfate transport system. Studies performed by P´erezCasti˜neira et al. (1998) in the presence of different inhibitors strongly supported multiphasic kinetics for sulfate uptake in C. reinhardtii. In our experiments, both S-starved and unstarved cells of Scenedesmus acutus had to replenish the amount of intracellular sulfur. However, in S-sufficient cells sulfur content increased from ∼0.100 to ∼0.140 g S/106 cells within 24 h of recovery, while in S-replete cells it increased from 0.070 to 0.140 and 0.250 g S/106 in the wild-type and in the Cr-tolerant strain, respectively. It therefore seems that, after starvation, the two strains had a higher capacity for sulfate uptake, i.e. that S-starvation induced an increase of the activity of the mechanisms involved in sulfur uptake and assimilation in both strains. This can be ascribed to the de-repression of the uptake system caused by a lowered intracellular pool of negative metabolic regulators of sulfate transport system. According to Schmidt and Jager (1992) and Smith et al. (1997), sulfate uptake seems to be under a feedback regulation by intracellular sulfate pool or endproducts of sulfate uptake or sulfate assimilation. Herschbach and Rennenberg (1994) and Lappartient and Touraine (1996) suggested that glutathione is involved in the control of SO4 2− uptake in higher plants, the increase of ATP sulfurylase activity and SO4 2− uptake being correlated with a decrease in tissue GSH concentration. Smith et al. (1997) reported that the capacity to take up sulfate increased in barley roots following sulfur starvation and, upon re-supply of sulfate, decreased rapidly concomitantly with the rise in tissue sulfate, Cys and GSH content. Besides, Bolchi et al. (1999) provided evidence “for the glutathione-independent involvement of cysteine as a stereoscopic pretranslational modulator of the expression of sulfur status-responsive genes” in maize. The authors suggested a regulatory model which can allow plants to distinguish between transient variations of GSH levels and a sulfur shortage with compromised protein synthesis. Actually, we observed a drastic reduction in Cys and GSH pools in the two strains of S. acutus when subjected to 7 days of S-starvation, a condition that may have triggered the up-regulation of sulfate uptake/assimilatory process, thus leading to the increased rate of Cys biosynthesis upon sulfate re-supply. The up-regulation of sulfate uptake mechanism following S-starvation can explain the higher tolerance to Cr(VI) displayed by S-starved strains of S. acutus and is consistent with the transient character of the increased tolerance. Since during 24 h of recovery after S-starvation the wild-type
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accumulated a significantly lower amount of sulfur than the Crtolerant strain, it can be hypothesized that the tolerant strain has higher activity/affinity enzymes and/or that the down-regulation of the sulfate uptake/assimilatory process in the wild-type occurs at lower levels of the negative metabolic regulators than in the Crtolerant strain. The latter hypothesis implies that the Cr-tolerant strain is more sensitive to the decrease in the intracellular endproducts of sulfate assimilation processes. It is also consistent with the higher levels of Cys and GSH observed in the stock culture compared to the wild-type strain, as well as the higher level of Cys in S-replete cells. Moreover, it seems to be supported by the rate of increase in free Cys and GSH pools observed in S-replete and S-sufficient cells during the first hour of recovery. Actually, GSH synthesis rate in S-replete cells of the wild-type was almost 50% higher than in S-sufficient cells, while in the Cr-tolerant strain it was in both cases essentially the same and higher than in the wild-type. This suggests that, in the Cr-tolerant strain, even the reduction of GSH pool suffered during 7 days in standard medium is sufficient to induce the increase in GSH synthesis rate to the probably highest value (2.0 mol SH/g d.w. per hour). Considering the levels of both free Cys and GSH, the rate of increase was much higher in S-replete than in S-sufficient cells in both strains and in the Cr-tolerant strain than in the wild type. This different behaviour can explain the differences in tolerance between the two strains despite their similar GSH content just after starvation and during 48 h recovery. These findings in the unicellular alga S. acutus seem to be in agreement with the regulatory model suggested by Bolchi et al. (1999) for higher plants. Higher sensitivity to the decrease in the levels of intracellular end-products of sulfate assimilation processes leading to an earlier up-regulation could thus be regarded as a primary reason of the tolerance to chromium in the selected strain of S. acutus.
5. Conclusions Sulfur-starved cells of the wild-type and Cr-tolerant strains of Scenedesmus acutus showed a higher tolerance to Cr(VI) than unstarved cells. The increased tolerance observed upon sulfate re-supply was due to physiological transient modifications ascribable to phenotypic plasticity and can be regarded as a consequence of specific responses of the algae to sulfur deprivation. Actually, after starvation, the two strains had a higher capacity for sulfur uptake and were able to rapidly restore GSH pool and increase free Cys to levels almost twice those of unstarved cells. These responses suggest that the higher tolerance to Cr(VI) after S-starvation is linked to the up-regulation of the sulfate uptake/assimilatory process. The greater sulfur uptake and rate of increase in free Cys and GSH content after starvation suggest that the Cr-tolerant strain may have a higher sensitivity to the decrease in the levels of intracellular end-products of sulfate assimilation processes than the wild-type. This can lead to an earlier up-regulation of these processes and could constitute a primary reason of the tolerance to chromium in the selected strain of S. acutus.
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