Elsevier

Aquatic Toxicology

Volume 68, Issue 4, 14 July 2004, Pages 315-323
Aquatic Toxicology

Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae)

https://doi.org/10.1016/j.aquatox.2004.03.020Get rights and content

Abstract

Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 μM). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one.

Introduction

Many algal species can survive in heavy metal-polluted environments by means of internal and/or external detoxification mechanisms (Pawlik-Skowronska and Skowronski, 2001). It is a well-known fact that algae can react to heavy metal stress by synthesizing compounds which can chelate toxic metals, namely reduced glutathione (GSH)—the most important non-enzymatic antioxidant, acting also as free-radicals scavenger (Nagalakshmi and Prasad, 2001)—and phytochelatins (PCs) (Gekeler et al., 1988), low molecular weight thiol peptides derived from GSH, and having structure (γGlu-Cys)n-Gly, with n=2–11 (Grill et al., 1985). PCs are synthesized in the cytosol by the constitutive enzyme phytochelatin synthase after its heavy metal-dependent activation (Grill et al., 1989). PCs are involved in primary detoxification mechanisms in both lower and higher plants (Cobbett, 2000, Sanita di Toppi et al., 2002, Sanita di Toppi et al., 2003, Cobbett and Goldsbrough, 2002) and may play a relevant role in the homeostasis of essential metals (Grill et al., 1988).

Some species and ecotypes of algae can live in the presence of toxic metal concentrations that are lethal for other species or populations (Torres et al., 1997, Knauer et al., 1998, Skowronski et al., 1998, Morelli and Scarano, 2001, Pawlik-Skowronska, 2000, Sanita di Toppi et al., 2003). Many authors have referred to this capacity as resistance or tolerance (Baker, 1987). On the basis of the growth rate in the presence of Cd, Chlorella vulgaris, C. saccharophila, Skeletonema costatum, Asterionella formosa and Selenastrum capricornutum (currently named Pseudokirchneriella subcapitata) can be grouped among the most sensitive species, while Scenedesmus obliquus, Ankistrodesmus falcatus, Navicula incerta, Asparagotsis armata, Tetraselmis gracilis and Phaeodactylum tricornutum can be considered more tolerant (Torres et al., 1997). In the present study, tolerance is considered “the heritable increase in the ability to cope with excessive metal, naturally or artificially selected under the pressure of toxic levels of metal exposure” (Ernst et al., 1992). Several authors suggest that tolerance sensu stricto can be achieved by means of adaptive mechanisms, possibly linked to constitutive detoxification mechanisms (Baker, 1987, Sanita di Toppi and Gabbrielli, 1999, Hall, 2002). However, tolerance mechanisms to heavy metals in algae are not completely clear as yet (Pawlik-Skowronska and Skowronski, 2001). Torres et al. (1997) suggested that the high tolerance of Phaeodactylum tricornutum can be due not only to the increase in PC production, but also to the increase in molecular length of thiol peptides compared to other species such as Chlorella fusca and Stichococcus bacillaris. Moreover, Perez-Rama et al. (2001) concluded that Tetraselmis suecica would be one of the most tolerant microalgae of Cd, since it is able to synthesize longer PCs than other species.

It is a well-known fact that different species have different environmental requirements for optimal growth and that many environmental factors (such as pH, salinity, free ions, organic and inorganic ligands) can influence metal uptake and toxicity. The availability of algal strains having identical requirements for growth but different sensitivity to heavy metals could help to understand mechanisms underlying tolerance of heavy metals, by comparing their biochemical responses to metal exposure. In our laboratory, a chromium-tolerant strain of the freshwater unicellular green alga Scenedesmus acutus (Chlorophyceae) was selected by treating the wild type population with sublethal concentrations of hexavalent chromium [Cr(VI)] for three months (Corradi et al., 1995). Tolerance was maintained even after culture in Cr(VI)-free medium, suggesting that the acquired capacity of the tolerant strain to cope with high concentrations of Cr(VI) is heritable. The Cr-tolerant strain was compared with the original population for sensitivity to copper (Cu) and zinc (Zn); it proved to be co-tolerant of Cu too, and, moderately, of Zn (Morsi Abd-El-Monem et al., 1998).

In the present study these two algal strains (wild type and Cr-tolerant) were taken as a model for the analysis of detoxification and tolerance mechanisms of Cd stress as well. GSH (as free radical-scavenger and precursor of PC synthesis), cysteine (Cys, the main limiting substrate for GSH biosynthesis) (Noctor et al., 1996) and PCs can be involved in these mechanisms. Cr(VI) and Cu have proven to be inducers of free oxygen radicals (Kotas and Stasicka, 2000, Nagalakshmi and Prasad, 2001); Cu also stimulates the biosynthesis of PCs, with consequent depletion of the glutathione pool. Since Cd detoxification mechanisms also rely on similar processes, we hypothesize that the Cr-tolerant strain of S. acutus could be also co-tolerant to Cd. We further hypothesize that, in the case of differences in sensitivity to Cd between the two strains, this could depend on their different ability in Cys, GSH and PC biosynthesis. In order to verify the above mentioned hypotheses, we first studied the relative sensitivity of the wild type and of the Cr-tolerant strain to Cd by means of growth and recovery tests. Afterwards, we compared the production of Cys and non-protein thiol compounds (GSH and PCs) in these two strains, in the absence or presence of Cd.

Section snippets

Growth and recovery test

Differences in tolerance of Cd between the wild-type strain and the Cr-tolerant one were analysed by means of growth tests in the presence of various Cd concentrations and recovery tests. Synchronized axenic cultures of the two strains of Scenedesmus acutus, i.e. the wild-type and the Cr-tolerant, were grown in liquid culture medium (Chiaudani and Vighi, 1977) in a climate-controlled chamber, at 25±1°C, 230 μmol m−2 s−1 and 16-h photoperiod. The cells were counted after 3 days, when the algae were

Results

The growth experiments on the two different strains of Scenedesmus acutus exposed to a range of Cd concentrations (1.125, 2.25, 4.5, 9, 13.5 and 27 μM) revealed significant differences in sensitivity to Cd (Fig. 1). The toxic effects of Cd were clearly observed at 4.5 μM Cd; after a 7 day-exposure, the growth of the wild type strain was reduced by 82% in comparison with control cultures, and the growth of the Cr-tolerant strain by approximately 58%. In the presence of low Cd concentrations (1.125

Discussion

Two strains of Scenedesmus acutus with different Cr tolerance were studied to investigate their co-tolerance of Cd. The Cr-tolerant strain of S. acutus proved to be co-tolerant of Cd, as evidenced by the growth experiments and the recovery tests after exposure to high Cd concentrations that appeared to be lethal for the wild type strain. The tolerant strain was able to recover better than the wild type one even after exposure to 27 μM Cd, which is a toxic concentration to green microalgae (

Conclusion

A different sensitivity to Cd in the wild type and in the Cr-tolerant strain of S. acutus was demonstrated by means of growth and recovery tests. Co-tolerance of Cd in the Cr-tolerant strain can be achieved by means of elevated Cys amounts, rather than high levels of GSH and PCs; this is true especially at Cd concentrations ≤9 μM. At higher Cd concentrations, the response to Cd in this strain appears to rely on both elevated levels of Cys and an increased biosynthesis of GSH and PCs, compared

Acknowledgements

We wish to thank Prof. Tadeusz Skowronski for his precious comments and suggestions. We are also grateful to Corrado Zanni for his excellent technical assistance.

References (39)

  • L. Sanità di Toppi et al.

    Response to cadmium in higher plants

    Environ. Exp. Bot.

    (1999)
  • E. Torres et al.

    Long-chain class III metallothioneins as a mechanisms of cadmium tolerance in the marine diatom Phaedactilum tricornutumBohlin

    Aquat. Toxicol.

    (1997)
  • A.J.M. Baker

    Metal tolerance

    New Phytol.

    (1987)
  • G. Chiaudani et al.

    Applicazione di un saggio algale standard per lo studio di fenomeni di tossicità

    Nuovi Ann. Ig. Microbiol.

    (1977)
  • C.S. Cobbett

    Phytochelatins and their roles in heavy metal detoxification

    Plant Physiol.

    (2000)
  • C.S. Cobbett et al.

    Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis

    Annu. Rev. Plant Biol.

    (2002)
  • J.A. De Knecht et al.

    Phytochelatins in cadmium-sensitive and cadmium-tolerant Silene vulgaris: chain length distribution and sulphide incorporation

    Plant Physiol.

    (1994)
  • C.H.R. De Vos et al.

    Glutathione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in Silene cucubalus

    Plant Physiol.

    (1992)
  • W.H.O. Ernst et al.

    Metal tolerance in plants

    Acta Bot. Neerl.

    (1992)
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