Biological and chemical effects of cadmium on "Chlamydomonas reinhardtii"

Thesis

Reference

Biological and chemical effects of cadmium on "Chlamydomonas reinhardtii"

SIMON, Dana Florina

Abstract

La toxicité du cadmium sur un organisme est dépendante de sa biodisponibilité qui varie en fonction de la physicochimie du milieu aquatique. Le but de cette recherche a été de relier l'effet biologique à la biodisponibilité du cadmium en étudiant les interactions du métal dans la cellule au niveau génomique. Les techniques de détection différentielle (differential display) et biopuces ont été employées et dix gènes ont retenu l'attention comme biomarqueurs potentiels pour le cadmium. Ces gènes ont été revalidés pour des concentrations de cadmium variant entre 10[puissance -9] et 10[puissance -6] M. L'étude sur la toxicité du cadmium menée sur le terrain a démontré que les algues restaient vivantes au cours de leur exposition en milieu naturel et étaient affectées au niveau de l'efficacité photosynthétique. Certains des biomarqueurs sélectionnés dans la première partie de ce projet ont été activés durant l'exposition sur le terrain.

SIMON, Dana Florina. Biological and chemical effects of cadmium on "Chlamydomonas reinhardtii". Thèse de doctorat : Univ. Genève, 2008, no. Sc. 4037

URN : urn:nbn:ch:unige-40866

DOI : 10.13097/archive-ouverte/unige:4086

Available at:

http://archive-ouverte.unige.ch/unige:4086

Disclaimer: layout of this document may differ from the published version.

UNIVERSITÉ DE GENÈVE Section de chimie et biochimie Département de chimie minérale, analytique et appliquée

FACULTÉ DES SCIENCES Professeur Jacques Buffle

UNIVERSITÉ DE MONTREAL Département de chimie

FACULTÉ DES ARTS ET DES SCIENCES Professeur Kevin Wilkinson

Biological and Chemical Effects of Cadmium on Chlamydomonas reinhardtii

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention interdisciplinaire

par

Dana Florina SIMON de

Roumanie

Thèse No 4037

GENÈVE

Atelier de reproduction de la Section de physique

UNIVERSITÉ DE GENÈVE

FACULTE DES SCIENCES

Docforaf ès sciences mention interdisciplinaire

Thèse de

Madame Dana Florina S I M O N

intitulée :

" Biological and Chemical Effects of Cadmium

on Chlamydomonas reinhardfii "

La Faculté des sciences, sur le préavis de Messieurs J. BUFFLE, professeur honoraire et directeur de thèse (Département de chimie minérale, analytique et appliquée), K. J. WILKINSON, docteur et CO-directeur de thèse (Université de Montréal - Département de chimie

-

Montréal (QC), Canada), W. ZERGES, professeur associé (Concordia University

-

Biology Departement

-

Montréal (QC), Canada), et P. DESCOMBES, docteur (National Center of Competence in Research (( Frontiers in Genetics )), Département de zoologie et biologie animale), autorise l'impression de la présente thèse, sans exprimer d'opinion sur les propositions qui y sont énoncées.

Genève, le 20 octobre 2008

Thèse - 4037 -

N.B.- La thèse doit porter la déclara'tion précédente et remplir les conditions énumérées dans les "Informations relatives aux thèses de doctorat à l'université de Genève".

TABLE OF CONTENTS

RESUME DE LA THESE iii

I. INTRODUCTION 1

1.1. Biological aspects of the interaction between metals and organisms 2 1.2 Intracellular binding and sequestration 4

1.3 Compartmentalization 7

1.4 Overview of genes induced by cadmium 9

II. METHODS USED FOR GLOBAL TRANSCRIPTOME ANALYSIS AND

QUANTIFICATION OF EXPRESSION LEVELS 15 2.1. Review of methods available for global transcriptome analysis 15 2.2. Microarray analysis 16 2.3. Differential display 18 2.4. Real time quantitative Polymerase Chain Reaction (qPCR) 20 2.5. Photosynthesis efficiency 22 III. CHEMICAL ASPECTS OF INTERACTION BETWEEN METALS AND

ORGANISMS 26 3.1. Speciation of metals in aquatic systems 26 3.2. Physicochemical conditions that favors metal uptake 27 3.3 Models describing biouptake 30

IV. RESEARCH FRAMEWORK AND OBJECTIVES 31 V. GLOBAL EXPRESSION PROFILING OF CHLAMYDOMONAS REINHARDTII EXPOSED TO TRACE LEVELS OF FREE CADMIUM 33

5.1 Introduction 33 5.2 Material and methods 34

5.2.1 Culture and exposure media 34

5.2.2. Differential display 34 5.2.3. Microarray analysis 36 5.2.4 Quantitative real time polymerase chain reaction (qPCR) 37

5.2.5. Bioaccumulation experiments 38 5.3. Results 39

5.3.1 Cadmium bioaccumulation 39 5.3.2. Microarray analysis revealed 12 genes 39

5.3.3. Differential display analysis revealed 23 up regulated genes 40

5.3.4 Cd-responsive genes as biomarkers for cadmium risk 45

5.4. Discussion 46

VI. CHEMICAL ASPECTS INFLUENCING METAL BIOUPTAKE AND

EXPRESSION LEVELS: CALCIUM AND MAGNESIUM EFFECTS 53 6.1 Introduction 53 6.2. Material and methods 56 6.3. Results and Discussion 59

6.3.1 Cd bioaccumulation as a function of variations in Ca and Mg 59 6.3.2. Responses of the cadmium biomarkers as a function of variations in Ca and Mg 60

6.3.4. Proton ion effect 63 6.4. Conclusions 66

VII. CADMIUM BIOMARKERS TESTED IN FIELD STUDIES ON THE LOT

RIVER, FRANCE 67 (Results of the 2007 and 2008 field campaigns) 67 7.1 Introduction 67 7.2 Material and methods 68

7.2.1. Algal preparation and exposure 70

7.2.2. Cellular viability 72 7.2.3. Bioaccumulation 73 7.2.4. Photosynthesis efficiency 74

7.2.5 RNA extraction and quantification 74 7.3 Results and discussion 75

7.3.1. Cell viability during the exposure 75 7.3.2. Photosynthesis efficiency analysis (PEA) 76

Laboratory PEA results 76 PEA 2007 campaign field results 78

PEA 2008 campaign field results 80 7.3.3. Bioaccumulation of Cd in situ (2007) 83

Internalized metal concentrations (2007) 84 7.3.5 Cd biomarkers response as a function of internalized Cd (2007) 88

7.3.6 Cadmium biomarkers (2008) 89

7.3.7 Discussion 92 7.4. Conclusions 95

VIII. OVERALL CONCLUSIONS AND FUTURE PERSPECTIVES 97 IX. BIBLIOGRAPHY 99 X. ANNEXES 113

RESUME DE LA THESE

Les métaux se retrouvent dans l’environnement, qui est un milieu très complexe, sous différentes formes. La concentration totale d’un métal dans le milieu aquatique ne suffit pas à décrire ses effets biologiques car les formes du métal ne sont pas également disponibles pour les organismes. En conséquence, la toxicité engendrée par un métal sur un organisme peut varier et est très dépendante de la physicochimie du milieu. La biodisponibilité d’un métal se résume par sa capacité de réagir avec les composantes de la cellule afin d’engendrer une réaction. Dans ce travail, le but de la recherche a été de relier l’effet biologique à la biodisponibilité du Cd.

Identification des meilleurs gènes biomarqueurs

Pour relier l’effet biologique et la biodisponibilité, l'étude des interactions du métal dans la cellule au niveau génomique s’est révélée une étape incontournable dans le projet. Dans le cadre de cette recherche, l’agent agressant était le cadmium, un métal non essentiel au développement et maintien des organismes. Par les techniques de détection différentielle (différentiel display) et biopuces, dix gènes ont retenu l’attention comme biomarqueurs potentiels pour le cadmium. En effet, on a constaté un accroissement de l’expression des gènes retenus en fonction de la concentration de cadmium libre présent dans le milieu d’exposition. Ces gènes ont été revalidés pour des concentrations variant entre 10^-9 et 10^-6 M par la méthode la plus sensible disponible pour la quantification de l’expression génétique:

l’amplification en chaîne par polymérase quantitative en temps réel.

Paramètres physicochimiques influençant la bioaccumulation du Cd

Dans le but de mieux prédire et comprendre les effets des variations des paramètres physicochimiques dans les conditions naturelles, la présence des compétiteurs Ca et Mg sur la bioaccumulation du Cd ainsi que la réponse des biomarqueurs ont été étudié. La concentration du Cd internalisé diminue quand la concentration du calcium augmente. L’internalisation du Cd ne semble pas être influencée par la présence croissante du Mg dans le milieu d’exposition. La réponse de gènes biomarqueurs ont été testé en présence de concentration croissantes de Cd a pH 7 et à pH 5.7. Pour des concentrations semblables de Cd²⁺,

l’internalisation était moindre à pH 5.7 qu'à pH 7. L'induction des biomarqueurs était plus marquée à pH 7 (ex. AOT4) due probablement au fait qu’il y avait plus de Cd internalisé.

Études faite dans le cadre des campagnes de terrain 2007-2008.

Une étude sur la toxicité du cadmium a été menée sur le terrain dans la région de l’estuaire de la Gironde au sud de la France. Il a été démontré que au cours de ces deux campagnes (i) les algues restaient vivantes au cours de leur exposition en milieu naturel; (ii) les algues étaient affectées au niveau de l’efficacité photosynthétique dans les deux rivières; (iii) un nombre des biomarqueurs sélectionnés dans la première partie de ce projet ont été activés durant l’exposition sur le terrain.

Dix gènes ont retenu l’attention pour des études afin de déterminer leur potentiel comme biomarqueurs. Ces gènes ont été testés en laboratoire sous différentes conditions physicochimiques (variation de la concentration de calcium, magnésium et pH) et ensuite dans des conditions naturelles sur le terrain. La réponse des biomarqueurs est encourageante quant à leur utilisation comme outils de détection pour le cadmium biodisponible sur le terrain. Néanmoins, le milieu naturel étant très complexe leur utilisation doit être faite en complément aux méthodes analytiques déjà disponibles pour les mesures de spéciation analytique des métaux pour une utilisation adéquate et appropriée.

I. INTRODUCTION

Water is essential to all living organisms. Since water resources are continuously recycled, its contamination, especially by persistent chemicals such as metals, can be of great concern.

Indeed, the concentrations of trace metals in water are mainly controlled by atmospheric deposition and the weathering processes of soils and bedrock. Increased industrialization has accentuated the importance of metals in aquatic ecosystems.

Cadmium is a by-product of the metal industry (e.g. zinc smelting) and is used in the production of Cd-Ni batteries, as pigment stabilizers or in the coating industry and production of fertilizers. The atmospheric deposition of cadmium is due mainly to forest fires, volcanic activity and the incineration of cadmium containing wastes. In aquatic systems, the dangers of cadmium are due to its toxicity at low exposures, its persistence, its high aqueous solubility and mobility, and its important bioavailability (Bertin and Averbeck, 2006; Clemens, 2006).

To evaluate the contamination of aquatic ecosystems, total or dissolved metal concentrations are most often measured and compared to a legal upper limit. However, several studies have demonstrated that not all metal species are bioavailable to the same extent and that toxicological impacts will vary according to the physicochemistry of the medium (Heijerick et al., 2005; Slaveykova and Wilkinson, 2002; Wilkinson and Buffle, 2004). Thus, over the past thirty years, much research has focused on the role(s) of trace metal speciation, particularly (i) on which species are more susceptible to be accumulated by organisms, (ii) on which chemical conditions promote bioaccumulation (presence of high concentration of the bioavailable form), (iii) on the mechanism(s) by which metals induce toxicity inside the cell and (iv) on the various defense mechanisms that cells use against metal toxicity (Clemens, 2006; Kong et al., 1995; Kovalchuk et al., 2005; Mitra et al., 1975; Mohamed Fahmy Gad- Rab et al., 2006; Perego and Howell, 1997; Robards and Worsfold, 1991; Sanita di Troppi and Gabrielli, 1999; Seregin and Ivanov, 2001).

One of the challenges of environmental chemistry and toxicology remains how to best relate the chemistry of metal exposures to the biology of the observed effects. Recently, it has become possible to quantify gene expression and determine how the chemistry of the medium

can modulate cadmium exposures. The focus of this dissertation is global gene expression profiling and quantification of expression levels in an aquatic organism

1.1. Biological aspects of the interaction between metals and organisms

Tolerance and toxicity mechanisms are often hard to distinguish since they depend on the metal, the organism, the dose, the exposure time and the physiological state of the organism prior to exposure. Tolerance might be classified as mechanisms that maintain homeostasis during adverse conditions. Toxicity involves non reversible changes caused by an agent in the organism.

All organisms are exposed to cadmium (and other trace metals) either due to natural or anthropogenic events. Cadmium is believed to be non-essential for microorganisms. One documented exception involves a marine algal species that, due to the scarcity of Zn in oceanic waters, uses Cd instead of Zn as a cofactor in carbonic anhydrase (Park et al., 2007).

Microorganisms generally possess a basal tolerance to toxic metal ions that allows them to maintain growth and survive. Nonetheless, when concentrations are too high, the cell will react on a physiological level. Extremely toxic metal concentrations are rarely found in natural environments except in the case of point source pollution disasters.

Physiological disturbances that can are most often detected in the case of trace metal exposures are the decrease of growth rates (Chaffei et al., 2004; Kong, 1995), and changes in cell size (Pena-Castro et al., 2004). Other targets of metal toxicity associated with exposures to trace metals have been related to photosynthesis, the chlorophyll synthesis (Faller et al., 2005; Plekhanov and Chemeris Iu, 2003), cellular ATP content and respiration levels (Belyaeva et al., 2004a; Neuberger-Cywiak et al., 2005; Sokolova et al., 2005), chlorosis (Mohanpuria et al., 2007), the appearance of vacuolar granules (Rauser, 1987) and cell death (Ortega-Villasante et al., 2005). As mentioned before, when these physiological disturbances are detected, very high pollution levels are generally present. In most case of sub acute metal exposures, physiological changes can not be detected. In those cases, it might be possible to evaluate trace metal exposures at a molecular level.

Metals can affect organisms at the cellular level through a variety of mechanisms such as: a) displacement of metal cofactors b) redox potential c) alteration of global expression levels.

a) Displacement of metal cofactors. Cd is known to displace metal cofactors (e.g. Zn²⁺

and Ca²⁺) from undefined protein targets by directly binding to amino acid residues including cysteine, glutamate, aspartate and histidine (Faller et al., 2005; Hartwig, 2001; Rollin-Genetet et al., 2004; Schutzendubel and Polle, 2002). Trace metals can also displace endogenous metal cofactors from signalling proteins and enzymes. For example, in yeast, the binding of silver to a copper-binding site can inhibit Cu, Zn-superoxide dismutase (Irihimovitch and Shapira, 2000). In plants, Cd²⁺ may interfere with intracellular Ca²⁺ signalling pathways, reviewed by Deckert, 2005 (Deckert, 2005) or nutrient uptake (Merchant et al., 2006). In addition, many biosynthetic enzymes are inhibited due to the interaction of metal ions with their thiol groups. For example, the protein tyrosine phosphatase, implicated in maintaining the phosphotyrosine levels in cells, has been shown to be inhibited by presence of Cu²⁺. The metal inactivates the protein by the oxidation of cysteine on the active site.

b) Pro-oxidant status increase. Unlike other trace metals (e.g. Cr, Cu), Cd does not produce reactive oxygen species (ROS) by the Fenton reaction. Nonetheless, metals such as cadmium and lead can enhance the cell's pro-oxidant status by reducing the antioxidant glutathione pool resulting by an activation of calcium dependant systems and a modification of iron mediated processes. For example, in C. reinhardtii, a cadmium-mediated increase in ROS leads to a decrease in the reduced/oxidized glutathione (GSH) ratio, which in turn signals the translational arrest of the Rubisco large subunit. Changes to iron mediated processes may result on chlorosis, which is often observed as a decrease to the green color of leaves and algae cultures.

c) Synthesis of biomolecules responding to Cd stress. Documented responses to Cd exposure have been described for a number of organisms. They involve: (i) the production of peptides that respond to oxidative stress (e.g. glutathione (GSH), proline); (ii) the synthesis of molecular chaperones (heat shock proteins) to cope with damaged and misfolded proteins;

(iii) the activation of membrane transporters to transport the metal into vacuole or other storage compartments; (iv) the production of enzymes that are involved with sulfate

assimilation and (v) the biosynthesis of sulphur (S) containing amino acids and phytochelatins (Bertin and Averbeck, 2006; Deckert, 2005).

Finally, acute exposure to high levels of Cd will most likely result in an indirect oxidative stress and the disturbance of the redox potential. This perturbation will lead to an increased activity of a number of antioxidant enzymes including catalases, peroxidises, ascorbate peroxidise (APX), and superoxide dismutase (SOD) (Mendez-Alvarez et al., 1999). Chronic Cd exposures may result in mutagenesis, loss of DNA integrity and cell death (Ahsan et al., 2007; Zhou and Qiu, 2004).

In order to protect themselves against cellular damage, organisms struggle to counteract the toxic effects of metals by a variety of mechanisms. These mechanisms are principally:

intracellular sequestration (Clemens, 2001; Clemens, 2006; Nishikawa and Tominaga, 2001;

Ortiz et al., 1995; Rosen, 2002), extracellular sequestration (Ahner et al., 1998; Macfie et al., 1994; Macfie and Welbourn, 2000), storage in subcellular compartments such as vacuoles (Nagel et al., 1996), and efflux (Lee et al., 1996; Rauser, 1995).

1.2 Intracellular binding and sequestration

Only a small fraction of the transition metals in cells are present as “free” hydrated ions.

Indeed, recent work with Saccharomyces cerevisiae indictes that virtually all intracellular Cu is bound to ligands implying that cellular transport involves ligand exchange reactions (Rae et al., 1999). Thus, metal accumulation is related to both the uptake capacity and intracellular binding (Clemens et al., 2002). Different classes of (poly)peptides such as glutathione, phytochelatins, metallothioneins (Schat et al., 2002), organic acids, amino acids (el-Enany and Issa, 2001; Oven et al., 2002; Rauser, 1999) and thioredoxins (Lemaire et al., 1999;

Lemaire et al., 2002) are involved in the protection of cells against metal ion damage (especially Cd, Cu and Hg). For example, in in vitro experiments, Cd complexes were 10 to 1000 times less inhibitory than the same concentration of Cd(NO3)2 for the enzymes rubisco, nitrate reductase, alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and urease (Kneer et al., 1992). The detoxification of other weakly sulphur binding trace metals such as Ni and Zn is more related to the presence of amino and hydroxyl groups. These

intracellular agents deserve attention because research indicates that they are the primary barrier against metal induced cell damage.

The tripeptide glutathione (GSH) is the major pool of cytosolic thiols occurring in concentrations in the range of 0.1-10 mM. Glutathione inside cells is present mainly in the reduced form (i.e. 500:1 reduced:oxidized) (Stryer, 1995). Changes in this ratio indicate a disturbance of the redox environment and have been observed to lead to increased expression of the genes GSH1 and GSH2 that are involved in glutathione biosynthesis (Ernst et al., 2008;

Rausch et al., 2007). These peptides might be the first barrier against oxidative damage by some metals (As, Cd, Cu, Zn, and Hg) by chelating these ions when they are in low concentrations. Decreased GSH levels have been observed following exposure to some toxic metals and prior to the synthesis of metallothioneins or phytochelatins (Mehra and Mulchandani, 1995). Nonetheless, the binding of GSH to Cd²⁺ is weak and the GSH pool cannot effectively contribute to a reduction in toxicity. Once the concentration of free metal ions is higher than the effective metal buffering capacity of the cell, the ratio of reduced:oxidized GSH changes and the cell activates other detoxification mechanisms (e.g.

production of phytochelatins or other chelating peptides).

PCs are (γ- Glu-Cys)n-Gly metal thiolate polypeptides that are synthesized from GSH by phytochelatin synthase (γ-glutamylcysteine dipeptidyl transpeptidase). Binding or sequestration by PCs has been shown to mediate metal detoxification within cells. For example, experiments with Thalassiosira weissflogii have shown that the Cd-PC complex functions to detoxify Cd (Lee et al., 1996). Phytochelatin production is induced very rapidly in this alga which then reduces Cd stress to basal levels. Phytochelatin synthase has been found in plants, yeast and algae (Al-Lahham et al., 1999; Klapheck et al., 1994; Vatamaniuk et al., 1999). Phytochelatin synthesis is promoted by the presence of different metals, although cadmium is believed to be the strongest inducer (Rauser, 1995; Steffens, 1990;

Zenk, 1996). Large variations of PCs production can be observed as synthesis depends on the available metal, its concentration and the organism. Morelli and coworkers (Morelli and Pratesi, 1997) have indicated that the synthesis of PCs is regulated by the internal cellular levels of trace metal. In their study, PCs were induced to the same extent for equivalent internal concentrations of Cd or Cu. In C. reinhardtii, 100 μM of cadmium in the bulk

solution induced PC production within 8 hours, as detected by HPLC. The same study revealed that As did not induce production at significant levels and that Hg was primarily bound to glutathione peptides (Howe and Merchant, 1992). Due their ubiquitous induction in a large number of eukaryotic and prokaryotic organisms, this peptide could presumably be used as a biomarker of trace metal stress (Gawel et al., 2001). Nonetheless, for A. thaliana that was modified to produce increased (+30%) levels of PCs, did not show the expected increased tolerance to Cd, but rather showed a higher sensitivity to Cd when high levels of essentials metals were included in the cultures. Such data may suggest that the high concentrations of PCs at very low (ambient) concentrations of essential trace metals could be due to the fact that the cellular uptake of some trace metals is enhanced when the concentration of essential metals are low, due to the fact that they share the same transporters (Sagman et al., 2003).

Metallothioneins (MTs) are ubiquitous low molecular weight, cysteine rich proteins that bind metal ions in thiolate clusters (Clemens, 2001). While it was previously believed that MTs were present only in animals cells, recent reports demonstrate that they are also present in plants (Liu et al., 2005). Nonetheless, MTs have not been found in C. reinhardtii but they do appear to be present in the marine alga, Fucus vesiculosus (Morris et al., 1999). With a bioremediation perspective in mind, Cai and coworkers (Cai et al., 1999) inserted a chicken MT gene into C. reinhardtii and determined that the transgenic algae had a slightly higher potential to grow in higher cadmium polluted media. Thus a MT gene seems to confer an increased resistance to cadmium.

Amino acids such as proline and cysteine also participate in Cd metal tolerance. The role of proline has been analyzed to see how it facilitates Cd detoxification in C. reinhardtii by the introduction of a gene Δ¹-pyrroline-5-carboxylate synthetase (P5CS) from the mothbean.

Increased proline levels enhanced protection in cells that were exposed to 100 μM Cd, not by sequestering the Cd, but by maintaining a more reducing environment (higher glutathione levels) in the cell. Sequestration by phytochelatins, rather than proline, was determined from extended X-ray absorption fine structure (EXAFS) spectroscopy. The ratio of reduced/oxidized glutathione was increased four fold while wild type cells showed a 70%

increase in malondialdehyde (a product of lipid peroxidation) levels, which is an indicator of

oxidative stress (Siripornadulsil et al., 2002). When exposed to cadmium, manganese and nickel, Scenedesmus armatus accumulated proline from the growth media; the presence of which minimized the toxic effects of these metals (el-Enany and Issa, 2001). In a similar manner, the metal toxicity in Chlorella vulgaris was largely reduced by amino acids including cysteine and histidine (Mohapatra et al., 1997). Furthermore, a drastic increase in histidine production was observed following Ni applications to A. lesbiacum (Krämer et al., 1996).

These data reinforce the observation that amino acids are important in metal chelation.

Citrate, malate and oxalate have been implicated in a range of processes including differential metal tolerance, metal transport trough the xylem and vacuolar metal sequestration. Citric acid has been hypothesized to be a major ligand at low Cd concentrations. For example, in Crotalari cobalticola, exposure to copper did not cause an increased production of phytochelatins although it did induce a 6.4 fold increase in concentrations of citric acid and a 31 fold increase in cysteine concentrations (Oven et al., 2002). The increase in cysteine was also observed in non accumulator plants such as R. serpentina and S. cucubalus. Experiments suggested that free amino acids were complexing the toxic trace metals in the cytosol and transferring the complexes into the vacuole where they would will ultimately be complexed by organic acids. This model was also proposed also for the hyperaccumulator, Thlaspi goesingense, where free histidine was thought to bind Ni in the cytoplasm (Krämer et al., 2000).

Thioredoxins (TRX) are small ubiquitous proteins of about 12 kD that are found in many organisms trough the phylogenic tree. They have highly reactive exposed disulfide sites on the conserved Cys-X-X-Cys active site. More than one isoform has been found and analyzed.

They are implicated in a wide range of biochemical pathways with their main role being a reducing agent. In many cells, reduced thioredoxins regulate the activities of enzymes by reducing disulfide to thiol.

1.3 Compartmentalization

Toxic metals are complexed in vacuoles under essentially three forms: phytochelatins, polyphosphate bodies and transformed products through glutathione conjugation.

Metal-phytochelatins complexes

It is generally assumed that low-molecular-weight Cd-complexes are formed in the cytosol prior to being transported into the vacuole where more cadmium and sulfide are incorporated in order to produce high-molecular-weight complexes (Clemens, 2001). HMT1, a protein with a similarity to the ABC-type transporter, is localized in the vacuolar membrane. It mediates the Mg-ATP energized transport of Cd-PC complexes and Apo-PCs in fission yeast (Ortiz et al., 1995). In S. cerevisiae, the ABC type transporter mediates Cd tolerance. This protein shares sequence homology with the human multi drug resistance associated protein (MRP) and was demonstrated to function as a glutathione S-conjugated pump. A cation diffusion facilitator, a metal transporter in bacteria, has also been identified in fungi, animals and plants. The main cations transported are Zn and Cd (Nies, 1992). The subcellular distribution of Cd was investigated in cell-wall defective mutants of C. reinhardtii. Ten percent of the Cd was in the cytosol and more than 50% was transferred into the chloroplast (Nagel et al., 1996).

Polyphosphate bodies

Polyphosphate bodies are linear polymers of orthophosphate that are found in the cells of all organisms. To date, there is a lack of evidence for any essential metabolic role except as a chelator of metal ions (Kornberg, 1995). Upon exposure to 20 μM Cd, 200 μM Cu and 1.5 mM Zn, ultrastructural changes in the cells of C. acidophila were evaluated by transmission electron micrography (TEM). Non-membranous, electron dense deposits were observed in the vacuoles. X-ray analysis demonstrated that the electron dense deposits contained both phosphate and Cd. Cells that recovered from Cd stress simultaneously increased the peaks of cytoplasmic and sugar phosphates (Nishikawa et al., 2003). In a study of the diatom Skeletonema costatum by ultrastructural analysis and Electron Energy Loss Spectroscopy (EELS), cells exposed to 1.8 μM Cd produced inclusions that were transferred into the vacuole. Exposure to 0.7 μM Cu was thought to be more toxic because it did not end up in the vacuoles via the inclusions to the same extent as the Cd (Nassiri et al., 1997).

Glutathione-S conjugated complexes

Glutathione S-transferases (GSTs) are most often thought of as detoxification enzymes since they catalyze the conjugation of glutathione to a variety of hydrophobic electrophilic and usually cytotoxic substrates. In phase I, a detoxification pathway is observed for electrophilic compounds in which enzymes such as cytochrome P450 introduce functional groups into the substrates. In phase II, enzymes such as UDP:glucosyl transferase and GST use the functional group as a site for further conjugation that usually results in a less toxic and more soluble compound. Finally, in phase III (compartmentalisation), ATP-dependent membrane pumps recognize and transfer the conjugates (e.g. herbicides and heavy metals) across membranes to be stored in vacuoles (Marrs, 1996). Transport is selective for oxidized glutathione-conjugates (Lu et al., 1997).

Efflux

C. reinhardtii contains vacuoles that are often filled with dense granules that are exocytosed. . C. vulgaris exude copper (Foster, 1977) while diatoms, coccolithophores and cyanobacteria exclude metal-complexes as a detoxification mechanism (Morel and Price, 2003). The excretion of metals (free or complexed) is also used ubiquitously by prokaryotic organisms (Nies, 1992).

Extracellular sequestration

The cell wall and biological membrane have important roles to play in regulating the toxicity of metals. The cell wall of plants including that of algae has the capacity to bind metal ions (Vogeler et al., 1990). For example, the cell wall of C. reinhardtii consists of several soluble glycoproteins, hydroxyproline, different types of sugars (arabinose, manose, galactose and glucose) and a number of insoluble glycoproteins. The wall-less strain is apparently more sensitive than the walled strain to Cd, Co, Cu and Ni indicating that the cell wall plays an important role in metal tolerance (Macfie et al., 1994). Nonetheless, Cd internalization was not significantly different among the wild type and cell wall-less strain (Kola, 2004).

1.4 Overview of genes induced by cadmium

Variations of global gene expression patterns and proteome composition have been described

responses, most of the studies in the literature have used high metal concentrations (100-500 μM Cd) and long exposure times (several days) (Chiang et al., 2006; Zhu and Thiele, 1996).

Some of the genes that have been identified following Cd exposures are discussed below:

Genes involved in sulphur assimilation

It has been established that the presence of Cd can stimulate the expression of S-transporters in order to ensure that sufficient sulphur levels are available for metal detoxification (Ernst et al., 2008).

Figure 1. Pathway for sulphate assimilation including enzymes that are up or down regulated upon exposure to cadmium (Ernst et al., 2008). Most of the enzymes are up-activated to favor sulphate (SO42-

) incorporation.

As illustrated in Fig. 1, the presence of cadmium generally stimulates high affinity S transporters (HAST) in order to ensure an increase of sulphate in the cell needed for Cd detoxification. Therefore, most of the enzymes involved in the assimilation of S and its transformation into S^2- pathway are stimulated positively by presence of cadmium (e.x.

HAST, ATPS, APR, SiR, SAT, PCs, GSHS). Therefore, the concentration of sulfur inside the cell is generally increased due to an oxidative stress produced by metal ions.

In C. reinhardtii, internal sulfur concentrations increased by 7.5% in the presence of cadmium (Mosulén et al., 2003). This was not surprising since sulphate is used for the production of

glutathione, cysteine, serine and methionine. Sulphate is incorporated into the skeleton of serine catalyzed by the cysteine synthase complex that involves two enzymatic components:

serine acetyl transferase (SAT) and O-acetyl-L-serine (thiol) lyase (OASTL). SAT catalyzes the acetylation of serine by acetyl CoA to form O-acetylserine (OAS) and OASTL catalyzes the conjugation of sulfide with OAS to form cysteine (Fig. 1). An increase in the activity of SAT and OASTL was shown to boost the production of cysteine (Dominguez et al., 2003).

Cysteine is well known to be amino acids of the peptide gluthatione (GSH) which is used for the synthesis of phytochelatins (PCs). Both sulphate starvation and metal exposure produced the same biochemical effects in C. reinhardtii. Since metal detoxification demands a significant amount of sulfur, mechanisms involving sulfur may be limited during algal adaptation to contaminated environments.

Genes coding for metal transporters

Genes known to be involved in cadmium tolerance are often transporters that either transport Cd-complexes into organelles or export the metal out of the cell. For example, the genes CAX2 and CAX4 encode Cd/H antiports that mediate the transfer of Ca²⁺ ions from the cytosol into vacuoles with a corresponding flux of H⁺ from vacuoles to the cytosol. Expression of the CAX2 and CAX4 genes from A. thaliana in Nicotiana tabacum resulted in vacuolar sequestration of Cd and an enhanced tolerance to Cd (Koren'kov et al., 2007). On the other hand, the gene encoding heavy metal ATPase (HMA4) in A. Thaliana was shown to participate in the tolerance mechanism in plants by expulsion of Cd/Zn in order to maintain low concentrations in the cytoplasm. This P1B-type ATPase was demonstrated to transport the metals ions against their electrochemical gradients using the energy provided by ATP hydrolysis (Courbot et al., 2007). Other studies demonstrate that transporters such as the Natural Resistance Associated Macrophage Protein (NRAMP) (Ouziad et al., 2005), cation diffusion facilator (CDF) transporters and a multidrug resistance-associated protein (MRP/GS-X) (Perego and Howell, 1997)(Paulsen and Saier, 1997) are important players in Cd toxicity since increased transcript levels of these genes have been observed under metal stress. In addition, CrMRP2 has been shown to be implicated in the formation /accumulation of stable high molecular weight protein complexes (HMW PC-Cd) in the vacuole (Wang and Dei, 2006). The screening of Cd responsive genes in A. thaliana has resulted in the identification of a gene that encodes a putative metal binding protein, CdI19, which upon

introduction into yeast cells has conferred an increased tolerance to cadmium exposure (Suzuki et al., 2002). This protein is exclusively located at the plasma membrane and it seems to have an important role in the maintenance of heavy metal tolerance and detoxification by conferring the plasma membrane with the capacity to serve as an initial barrier against the influence of free metal ions into the cells (Suzuki et al., 2002). C. reinhardtii has few families of transporters that are known to be involved in metal homeostasis and detoxification. For example, it contains five types of CDF, two HMA and three types of NRAMP transporters.

Recently, Hanikenne and coworkers (Hanikenne et al., 2005b) have shown that CrCds (a homolog of the mitochondrial, heavy metal transporter (HMA)) can play an essential role in cadmium tolerance, possibly by exporting Cd out of the mitochondria.

Genes coding for heat shock proteins (HSP)

In studies of Cd exposure, an additional family of genes encoding for the heat shock proteins (HSP) frequently appear to be induced. Weber et al., 2006 (Weber et al., 2006) compared the transcriptome response of A. thaliana (metal tolerant species) to A. halleri (metal resistant species) following a Cd exposure. In addition to genes involved in sulphate assimilation and in the GSH pool, genes that coded for HSPs proteins were induced (Koizumi and Yamada, 2003). HSPs are proteins induced by abiotic stress. Their constant apparition in Cd exposure studies indicates that this family has an important role in Cd tolerance. For example, Blechinger et al. (2002) (Blechinger et al., 2002) assessed hsp70 gene activation as a measure of cadmium toxicity in zebrafish. They also produced an hsp70—enhanced green fluorescent protein (eGFP) reporter gene and demonstrated that this reporter gene was an accurate and reproducible indicator of cadmium in the range of 0 to 125 μM Cd. Concentrations below 0.2 Cd μM produced no effect.

The genome of the eukaryotic green alga C. reinhardtii encodes a battery of metal transporters, one of which has been shown to be required for Cd tolerance (Hanikenne et al., 2005a; Wang and Wu, 2006). Differential display (Rubinelli et al., 2002) has been used to identify 21 genes that were induced by a 2 h exposure to 25 μM Cd. One of the most interesting Cd responsive genes found in their study (H43) was assessed by homology to a high CO₂ and iron depletion inducible protein in Chloorococcum littorale. This gene was induced following iron depletion and/or Cd exposure in C. reinhardtii wall-less mutants but

not in the wild type. Hanawa and co workers (Hanawa et al., 2007) concluded that H43 was induced both at the transcriptional and translational level under high CO2 conditions. Finally, a recent proteomic approach revealed that 150 μM of Cd enhanced the levels of numerous proteins, most notably proteins with antioxidant properties and those responding to misfolded proteins that arise under many different stress conditions (Gillet et al., 2006).

In C. reinhardtii, it has been demonstrated that the chloroplastic isoforms TRXh and TRXm are induced by heavy metals (Lemaire et al., 2003; Lemaire et al., 1999). Furthermore, the expression of both genes was shown to be regulated by Cd and Hg. At the mRNA level, a four fold induction was observed when cells were exposed to 100 μM Cd and a six fold induction was observed when cells were exposed to 1 μM Hg. The response of TRXh was faster (1 hour) than TRXm (2 hours). The stress induced by the trace metals might be related to the oxidative stress of proteins and amino acids. At first view, the induction of the TRXh and TRXm messenger levels could be an indirect effect linked to a modification of the redox state of the cell. However, when C. reinhardtii cells were tested with other inducers of oxidative stress (H2O2 and diamide), increased mRNA levels of the two TRX were not be observed, indicating that their induction in the presence of metals was not an indirect effect of oxidative stress. Analysis of the TRXh promoter revealed the presence of a tandem repeat as-1-related element that was not found in the TRXm gene. This element has been observed in parA and pGH2/4 genes that are regulated by auxin and Cd in tobacco cultures. The parA gene is expressed in the presence of Cd but does not respond to other plant hormones or heat shock (Kusaba et al., 1996). This specificity to Cd makes this gene interesting for further studies.

Table 1. Presentation of several important proteins encoded by genes involved in metal stress.

This is not an exhaustive compilation.

Protein Function Reference

ATPS, APR, SiR, SAT PCs, GSHS

Induced during sulphur requirement

Ernst et al., 2008 CAX2, CAX4 Cd/H antiporter Korenk’kov et al., 2005

HMA4 Protein function in expulsion

of Cd/Zn out of the cell

Cournot et al., 2007 P1B-type ATPase, NRAMP,

CDF, MRP/GS-X, CrMrp2, CrCds

Various transporters involved in the transport of Cd-

complex out of the cytoplasm

Ouziad et al., 2005 Perego et al., 1997 Paulsen et al., 1997 Wang et al., 2006

Cdl19 Block of Cd uptake Suzuki et al., 2002

HSPs Heat shock proteins induced

during abiotic stress

Weber et al., 2006, Blechinger et al., (2002) TRXh and TRXm Electron donors involved in

many biochemical reactions

Lemaire et al., 1999, 2003

II. METHODS USED FOR GLOBAL TRANSCRIPTOME ANALYSIS AND QUANTIFICATION OF EXPRESSION LEVELS

2.1. Review of methods available for global transcriptome analysis

Investigations of changes in gene expression induced by trace metals combined with the analysis of trace metal speciation are powerful tools to understand trace metal impacts. These interactions can be followed at the proteomic (i.e. protein production) or transcriptome levels (i.e. gene expression). In proteomic studies, many proteins can interact with trace metals due to their complexation by a number of amino acids. On the other hand, changes in gene expression are often the result of interactions between environmental stimuli and the gene products that underlie the regulation of the physiological responses.

Measurement of gene expression level

Genes are expressed by the process of transcription in which information from a gene is made into a functional gene product (mRNA). The mRNA is then translated into a protein. Under various physicochemical conditions, the mRNA level of a specific gene can be altered, resulting in an increase or decrease in its concentration. This is due to the fact that the cell needs a different amount of the protein encoded by this gene. The gene expression intensity is therefore assessed by the quantification of mRNA present in the system. Expression levels of a given gene are measured relative to a control gene whose expression level is stable under the tested conditions. By assuming that the expression level of the control gene corresponds arbitrarily to 1 fold induction, genes with higher than 1 fold induction can be identified.

By quantifying the steady state levels of specific messenger RNA (mRNA) of differentially expressed genes, it is possible to assess changes produced in the cell under specific external conditions. Several methods are available that provide the means to assess genome-wide expression after the exposure of an organism to different physicochemical conditions (Table 2). They can be divided into four categories: 1) microarray analysis; 2) differential display (DD, ADDER, SABRE); 3) subtractive hybridization (SH, SSH), 4) a mix of differential display and subtractive hybridization (RSDD, IPTG) and 5) serial gene analysis (SAGE).

Each of the methods has its own advantages and disadvantages. The main considerations are the time of analysis (e.g. microarray analysis: few days; differential display: weeks), the

number of samples that can be examined at once and the cost of the experiment (microarray is generally more expensive than differential display). Several of the important methods are described in more detail below.

Table 2. Presentation of main advantages and disadvantages of the methods used to assess global transcriptome analysis. This is not an exhaustive compilation of all methods.

Advantages Disadvantages Microarray¹ Gene function known.

Fast method relative to others.

Not available for all organisms. Limited to the number of genes indexed on the slide.

DD² ADDER³ SABRE⁴

Avoids loss of low abundant gene copies. Possibility to find novel genes.

Does not provide sequence information directly. Known to give false positives.

SH⁵ SSH⁶

Removes abundant mRNA.

Normalize for genes expressed at low levels.

Does not provide sequence information.

High number of PCR steps can result in false positives.

IPTG⁷ RSDD⁸

Mix of DD and SH to increase the efficiency of genome wide

identification.

Labor intensive.

SAGE⁹ Can be use to identify and quantitate new genes.

Technique is limited to laboratories with high throughput sequencing facilities.

1 Microarray (Barrett and Kawasaki, 2003). ²DD, Differential Display (Stein and Liang, 2002). ³ADDER, Amplification of Double-stranded cDNA End Restriction fragments (Kornmann et al., 2001). ⁴SABRE, Selective Amplification via Biotin and Restriction- mediated Enrichment (Schibler et al., 2001) ⁵SH, Subtractive Hybridization (Lee et al., 1991).

6SSH, Suppressive Subtractive Hybridization (Brown et al., 2004). ⁷IPTG, Integrated Procedure for Gene Identification (Wang and Rowley, 1998). ⁸RSDD, Reciprocal Subtraction Differential RNA Display (Dong-Chul et al., 1998). ⁹SAGE, Serial Analysis of Gene Expression (Liang, 2002).

2.2. Microarray analysis

DNA microarray technology has revolutionized genome analysis. Indeed, the number of publications that employ this method has increased exponentially since its commercialization at the end of nineties. DNA microarray technology is divided into two types of platforms:

spotted and oligonucleotide arrays. For the spotted arrays, the spots are cDNA, (e.g.

Affymetrix technology, GeneChip^® Arays) or small PCR (polymerase chain reaction)

fragments. The synthesized probes are deposited into surfaces (usually glass) in an ordered manner to form a grid. These arrays can be manufactured in genomic platform and usually exist for organisms where the genome was recently sequenced (e.g. C. reinhardtii). On the other hand, for oligonucleotide arrays, probes designed to match the open reading frames (exons) are directly synthesized directly onto surface. Short and long oligonucleotide arrays are prepared by synthesis on the plate (Barrett and Kawasaki, 2003; Mah et al., 2003; Watson et al., 1998). These arrays are commercialized by Agilent and Affymetrix and exist for most common model organisms used in research (e.g, human, mouse, yeast and recently numerous other model organisms).

In the spotted arrays, control and fluorescently (or radioactively) labelled cDNAs that are extracted from the sample of interest are hybridized to a library of cDNA that reflects the genome of a given organism and that has been spotted or synthesized directly onto a solid support. In each case, the probes molecules (cDNA, oligonucleotides) are derived from mRNA samples that have been cloned individually, amplified by PCR and printed on glass slides using robotic technology. The fluorescence or radioactivity that is incorporated in the target molecules can then be quantified in each microspot. Increased or decreased signals are indicative of up or down regulation (Fig. 2). For reference C. reinhardtii spotted microarrays were used in this research.

Control Sample

Reverse transcription and labeling

Labeled cDNA total RNA isolation

Competitive hybridization to the microarray slide

(- metal ) (+ metal)

Figure 2. Schematic representation of spotted microarray analysis

2.3. Differential display

The ADDER (Amplification of double stranded cDNA end restriction fragments) technique is a variation of differential display RT-PCR. It involves the construction and amplification of double stranded cDNA fragments that are complementary to the 3’-terminal moieties of mRNA. Tagged fragments can be differentially displayed to reveal genes that are up or down regulated. This method is highly sensitive, well adapted for genomes that have not been completely sequenced or for organisms with no commercial microarray available.

Figure 3. Schematic representation of ADDER analysis. (Kormann et al., 2001).

This variation of the differential display method (schematized in Fig. 3) consists in the isolation of mRNA from samples that have been exposed to metal-containing media and control samples (no metal). The mRNA is reverse transcribed (steps 1 and 2; Fig. 3) in a process that employs biotinylated oligonucleotide dT primers, which are primers that will anneal to the 3’end of the mRNA strand and contain a biotinylated end. The reverse transcribed strands are harvested from the reaction media by biotin-streptavidin interaction (streptavidin protein has a very strong affinity for biotin). Streptavidin is bound to magnetic

and the associated cDNA The cDNA is cut with restriction enzymes (Mbo1) in order to obtain pieces of cDNA with sizes that range between 50 and 400 base pairs and that can be easily amplified by PCR polymerisation (step 3). An adaptator is linked to the severed end of cDNA (step 4) and the streptavidin end is subsequently removed (step 5) using another restriction enzyme (Ascs1). This manipulation results in cDNA fragments that have ends with known sequences. This population of cDNAs is then amplified to obtain a larger quantity (step 6) using primers that are designed for the two ends of the cDNA section. Finally, a second PCR amplification is performed using 192 primers (combination of 16 - 5’ primers and 12 - 3’primers) (step 7). This step involves the amplification of the specific section between the adaptors, each section representing, in principle, a different gene. The amplification step is performed with a radiolabeled nucleotide (dCTP) that makes the PCR fragment radioactive and detectable by autoradiography following the separation of the fragments by gel electrophoresis. Due to the high sensitivity of the ADDER method, Kornmann and collaborators (Kornmann et al., 2001) showed that it was possible to reveal genes that were not previously discovered during the circadian gene expression profile in the liver. A shortcoming of this method is the low throughput analysis of the differentially expressed genes due to the fact that each fragment has to be cloned and sequenced to identify the differentially expressed genes.

2.4. Real time quantitative Polymerase Chain Reaction (qPCR)

There are a variety of methods for the quantification of mRNA: Northern blotting, dot blot assays, ribonuclease protection assays and real time qPCR. Real time qPCR is generally accepted as the most sensitive method, which can also discriminate closely related mRNAs.

For each of these methods, it is necessary to know the sequence of the gene of interest.

The polymerase chain reaction is a technique used to exponentially amplify a piece of DNA using an in vitro enzymatic replication. Using PCR, it is possible to amplify very few copies of DNA pieces over several orders of magnitude. Real time qPCR is a variation of the basic PCR technique that allows quantification of the PCR product over time. The schematization of the real time qPCR is presented in the Fig. 4. In order to perform the amplification, the mixture must contain:

- the DNA fragment to be amplified (represented by double strands)

- primers (represented by single short strands)

- TAQ polymerase (Thermus aquaticus polymerase which is the enzyme that replicate fragments during the amplification)

- deoxynucleoside triphosphate (dNTPs which are the building blocks from which the DNA polymerases synthesizes a new cDNA strand)

- SYBR Green fluorophore (represented by grey dots)

- and a buffer solution containing all the components used during the reaction Fluorescence is represented by undulating arrows.

Figure 4. Schematic representation of real time quantitative PCR analysis (Bustin, 2000).

The primers are nucleic acid strands that are complementary to the 3’ and the 5’ strand of the DNA. The reaction is carried out in a thermal cycler. The mixture goes through cycles of heating and cooling. The heating step (95 ºC) denatures the double stranded cDNA and allows

attachment of the primers at 60 ºC (steps A and B). A slightly higher temperature (the optimal temperature of TAQ polymerase is 70 ºC) allows amplification of the single stranded cDNA fragment with insertion of SYBR Green fluorophore (step C). The steps are repeated over 25 to 30 cycles. The increase in the amplified product is determined from the fluorescent detection of the SYBR Green fluorophore which emits only when it is inserted between double stranded DNA. The result is typically plotted as the fluorescence intensity as a function of the number of cycles (Fig. 5).

Figure 5. Plot of normalized fluorescence intensity (∆Rn) generated by the binding of the SYBR green dye as a function of the number of cycles (Ct) of the PCR reaction. The normalized fluorescence signal is presented as a ratio of the SYBR green fluorescence intensity over that of a passive reference dye, ROX (5-carboxy-X-rhodamine).The passive reference is an inert dye whose fluorescence does not change during the reaction and is used to normalize the well-to-well differences that may occur due to pipetting errors or instrument limitations.

2.5. Photosynthesis efficiency

The ability of a plant to carry out photosynthesis is dependent on a range of factors including stresses that are caused by environmental conditions. At room temperature, chlorophyll a fluorescence is almost exclusively emitted by photosystem II (PSII) and it can therefore serve

as an intrinsic probe of the fate of its excitation energy. Direct measurements of the fluorescence signal have been used to describe how photosynthetic organisms react under conditions that affect their photosynthesis apparatus (Strasser et al., 2004; Strasser et al., 2005)

Light energy is absorbed by chlorophylls (circles in Fig. 6) which are packed into antennas.

The resulting excitation energy passes from one chlorophyll molecule to another until it is trapped by a chlorophyll with particular properties in the reaction centre. In the reaction centre, the energy of the excited chlorophyll is converted into a charge separation that creates a reducing potential. Light energy trapped by chlorophyll is used to drive photosynthesis (photochemistry) and the excess light is dissipated in non photochemical quenching processes as heat emission and reemission of the adsorbed radiation at longer wavelengths as such as fluorescence (peak at 685 nm of, Fig. 6)

heat

fluorescence

photochemistry

Figure 6. Schematic representation of the chlorophyll pool capturing a photon and the subsequent electron transfer to the reaction centre of PSII with a potential emission of heat and fluorescence. (www.rz.uni-karlsruhe.de/lichtsammelbild.jpg)

The fluorescence yield of PSII is determined by the open or closed state of the reaction centre Plastoquinone are molecules involved in the electron transport chain in photosynthesis processes. They are implicated in the transport of protons H⁺ from the stroma of the chloroplast to the thylakoid lumen. If the QA in a reaction centre is reduced (QA^-) the reaction centre is closed and the chlorophyll fluorescence of the antenna is high (FM). In contrast,

when the QA is in oxidized state, the reaction centre is open the fluorescence of the antenna is quenched (Fo).

OJIP Test

The fluorescence transient obtained from illuminating dark adapted cells (known as the Kausky effect) can be divided in different phases called O-J-I-P (Schansker et al., 2006; Toth et al., 2005; Toth et al., 2007) The Photosynthesis Efficiency Analyzer (PEA, Hansatech Instruments Ltd.) is the able to record fluorescence intensity values of the first second upon illumination, revealing the kinetics of the fluorescence response (Fig. 7).

Figure 7. Schematic representation of chlorophyll fluorescence intensity as a function of time following the illumination of a dark adapted sample (Strasser et al., 2004).

The chlorophyll-fluorescence curve, (OJIP test) can be used to assess a plant’s physiological status under different environmental conditions (Strasser et al., 2004; Strasser et al., 2005). As mentioned above, the shape of the curve can be divided into a few steps (named O-J-I-P). The different phases of the curve have been attributed to variations of the redox states of PSII. The P phase corresponds to FM, the maximum fluorescence yield. The J and I steps are dominated by reduced and oxidized plastoquinones (QA and QA^-). The O step is dominated by oxidized plastoquinone (i.e. the minimal fluorescence intensity because the sample is dark adapted for a period of few minutes before the light pulse). Three parameters are generally analyzed during the experiment: (i) FV/FM, (ii) P.I. and the (iii) Area above the curve.

i) FV is the difference between the maximal fluorescence intensity (FM or P step) and the minimal fluorescence intensity (Fo or O step). The ratio Fv/FM indicates the maximum quantum efficiency of PSII primary photochemistry (Ohira et al., 2005). A decrease in Fv/FM ratio generally indicates a stress situation. For example in healthy leaves the ratio is ∼ 0.84.

(http://www.science.uts.edu.au/des/StaffPages/PeterRalph/fluorescence.html)

ii) The P.I. is the overall Performance Index, which is a measure of the photosynthetic performance of the cell, constructed in analogy to the Nernst equation. This describes the redox potential in physicochemistry which takes into account: (i) the density of the reaction centers, (ii) the efficiency of the light reaction and (iii) the efficiency of the dark reactions (Bueno et al., 2003). The photon flux is absorbed by antenna pigments (absorption flux) which create excited chlorophyll. Part of the excitation energy is dissipated, mainly as heat and partially emitted fluorescence. Another portion of the adsorption flux part is channeled to the reaction centre (Fig. 6). The P.I. is a calculation of the probability that the part of the excitation energy that is captured goes through to the reaction centre (RC) and can be converted to redox energy by reducing the electron acceptor QA to QA-. The QA- is then reoxidized to QA reducing the electron transport chain beyond QA- and ultimately leading to CO2 fixation.

iii) The complementary Area surface above the fluorescence curve between Fo and FM

is proportional to the pool size of electron transport acceptors, indicating the PSII capacity The pool size is thought to decrease when the cell is exposed to conditions of stress.

III. CHEMICAL ASPECTS OF INTERACTION BETWEEN METALS AND ORGANISMS

3.1. Speciation of metals in aquatic systems

In aquatic systems, the concentrations of metals vary according to their geographical localization. For example, Fig. 8 shows the range of total concentrations of the majors cations and trace metals that can be found in the open sea or freshwaters (Buffle and De Vitre, 1994).

The concentration of cadmium varies between 10^-12 M and 10^-9 M in open sea water and between 10^-10 M - 10^-8 M in freshwater systems.

Figure 8. Trace and major metal concentrations in open sea and freshwaters (Buffle and De Vitre, 1994).

Metals may be partitioned among a number of different forms or species. Indeed, in most natural systems, metals are rarely found as free ion (e.g. Cd²⁺) but more often in the form of trace metal complexes (CdCl^-, CdCl2, Cd(CO3-

). Metals may also form stable complexes by interacting with inorganic (e.g. CO32-

) and organic ligands (e.g. proteins, amino acids), particles (e.g. colloids, phytoplankton, bacteria). The complexation of metals is dependant on

physicochemical conditions such as pH, temperature, ionic strength redox potential, presence and concentration of complexing agents. Each of the complex species may behave differently with respect to their mobility, lability and bioavailability and thus natural systems are under constant change and practically never reach true equilibrium. Therefore, the study of metal speciation in natural environments is complicated and the elucidation of the relationship(s) between the different physicochemical forms of the metals and their reactivity, mobility and bioavailability is often difficult. In the laboratory, under well controlled conditions, we can predict the equilibrium concentrations of most metal species by using equilibrium modelling software (e.g. MINTEQA2; (Allison et al., 1999)). Nonetheless, accurate model predictions are difficult to do for natural waters due to the fact that it is very difficult to have the precise chemical composition (all inorganic and organic components) of natural waters, and the stability constants for all metal complexes.

3.2. Physicochemical conditions that favors metal uptake

As mentioned previously, the determination of trace metal speciation in a specific environment is important because not all metallic species are bioavailable to the organism to the same extent. The physicochemistry of the interactions of trace metals with a microorganism can be schematized in Fig. 9. In order to understand the biological effects that are induced by trace metals, it is important to understand the role of physicochemical processes on the biouptake or metal adsorption to the cell wall or biological membrane since this is the first step of any toxicological impact.

Diffusion

The metal generally diffuses through bulk solution to the biological membrane where it can be adsorbed prior to desorption or transport into the microorganism. The process of adsorption and internalization describes the metal bioaccumulation step. Inside the diffusion layer the metal can form complexes with hydrophilic ligands (Lh-M) or lipophilic ligands (LL- M). These ligands include organic ligands such as fulvic substances, humic substances, proteins or organic acids. The metal can also form complexes with strongly binding biological ligands, M-Lbio, the best known of which are the siderophores. As a result, the metal can attain the cell membrane as a complex (e.g. Lh-M, LL-M, Lbio-M) or as the free aquo ion (M). At the

level of the cell membrane, the metal might react with a sensitive site by adsorption which may or may not be followed by biological transport (internalization).

Figure 9. Interaction of metals with microorganism at the cellular level. Diffusion, complexation, adsorption and desorption are generally accepted as the main steps leading to metal internalization. Upon internalization, the metal can interact with ligands inside the cell (M-Lbio), initiate signals at the molecular level, be expulsed by efflux or secretion or be stored into vacuoles (Worms et al., 2006).

Transport

The membrane is composed of phospholipids, proteins, and polysaccharides; some of them containing ionisable groups that confer a (often negative) charge on the biological surface.

The metal can bind to both unspecific sites (e.g. carboxyl groups of the cell wall) and specific transport sites. The cell wall and cell membrane contain proteins, some of which are transporters. Metals can be internalized by carrier mediated transport, through protein channels and by passive diffusion. Metal speciation will have an important influence on the type of transport, i.e. lipophilic species such as HgCH3Cl species likely cross the membrane by passive diffusion while most other complexes of toxic metal (e.g. Cd, Ni, Pb, Cr) must dissociate to release the free metal which use the essential metal (Ca, Mg, Zn) transporters, to

cross the membrane via carrier mediated transport. It is thought that Cd enters the cell through Zn and Ca transporters (Gitan et al., 2003).

Internalization

The overall biouptake process depends on the rate-limiting step that is encountered during bioaccumulation. Internalization (Jint) is often assumed to be the slowest step in the overall reaction, i.e. Jint <<Jdiff,Jkin, Jkin'. Jint however depends on the internalization rate constants and the density of transport sites on the membrane. Several steps of the overall process are potentially rate-limiting:

- Diffusion of trace metal complexes to the biological surface (J_diff). Diffusion is slower when the metal is complexed to a macromolecule or colloidal ligand. Diffusion limitation (J_int > J_diff) is more frequently observed in marine rather than freshwater systems (Wilkinson and Buffle, 2004). It has been shown that in natural waters (contrary to laboratory conditions), the density of transport sites may be large and J_diff may become rate limiting. This situation arises when the transport system is stimulated in response to metal depletion, most likely for essential metals only (Hassler et al. 2003).

- Chemical reactivity (Jkin) of the trace metal in the bulk solution. The Jkin can become limiting (Jint > Jkin) in situations where complex (metal-ligand) lability is very low and the free metal concentration is too low. Some complexes are inert and dissociate very slowly: the metal is then “trapped” in the external solution.

- Chemical reactivity (Jkin') of the trace metal with transport sites at the cell membrane surface.

In either of these limiting cases (either J_diff or J_kin are limiting), bioavailability will be best predicted by a ponderated sum of free and labile species. Nonetheless, even though natural systems are practically never at equilibrium in the vicinity of the cell, it is often possible to assume a rate-limiting internalization that leads to steady-state conditions that can be predicted on the basis of equilibrium considerations.

In all cases, the physicochemistry of the bulk media surrounding the cell will have an important influence on the biouptake process. Furthermore, certain biological responses to the metal (e.g. efflux, pH changes at the cell surface, modification of surface charge) can also influence the physicochemistry of the surface, thus potentially modifying the internalization flux.

Not all organisms react in the same manner to trace metal exposures. In addition to the chemistry, the process of biouptake and metal sensitivity are dependent on biological factors such as the nutrient status and tolerance mechanisms. For example, for a decreased nutrient supply, the cell is more likely to be susceptible to metal toxicity because of a tendency to take up non essential trace metals at the same time as essential metals. In addition, differences may be observed between prokaryotes and eukaryotes since prokaryotes possess numerous systems that are designed to exclude bioaccumulated metal (Nies, 2003; Nies and Silver, 1995).

Eukaryotes generally reduce metal bioaccessibility by complexing the internalized metal, and then redistributing it among different compartments or vacuoles (Zenk, 1996; Zhou and Qiu, 2004).

3.3 Models describing biouptake

Most studies that examine the bioaccumulation of trace metals suggest that trace metal uptake is best related to the concentration of free metal in the bulk solution which has been interpreted to suggest that the metals in solution are in equilibrium with those at the surface of the biological organism and that equilibrium models based upon measurements of the free ion are likely the best predictors of trace metal bioaccumulation (Morell, 1983; Slaveykova and Wilkinson, 2005). Indeed, the FIAM (Campbell, 1995b; Morell, 1983) and BLM (Paquin et al., 2002) models have been developed to explain correlations between the concentration of free metal ions in solution and observed biological effects. For both models, metal uptake fluxes can be predicted from any of the metal species at equilibrium. In the case of the BLM and the FIAM, competitive effects are taken into account by predicting quantities of metal bound to site of action using equilibrium constants that have been determined from toxicity experiments.