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Environmental Research 105 (2007) 234–239

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Effects of chromium on photosynthetic and photoreceptive apparatus ofthe alga Chlamydomonas reinhardtii

M. Cecilia Rodrıgueza, Laura Barsantib, Vincenzo Passarellib, Valter Evangelistab,Visitacion Confortia, Paolo Gualtierib,�

aDepartmento de Biodiversidad y Biologıa Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,

Pabellon II, C1428EHA, Buenos Aires, ArgentinabIstituto di Biofisica, CNR, Via Moruzzi 1 56124, Pisa, Italy

Received 19 October 2006; received in revised form 15 January 2007; accepted 29 January 2007

Available online 7 March 2007

Abstract

Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr)

has become a serious pollutant in diverse environmental settings. The presence of Cr leads to the selection of algal populations able to

tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms include biosorption,

diminished accumulation, precipitation, reduction of Cr6+ to Cr3+, and chromate efflux. In this paper we describe the effects of Cr6+

(the more toxic species) on the photosynthetic and photoreceptive apparatus of the fresh water unicellular alga Chlamydomonas

reinhardtii. We measured the effect of the heavy metal by means of in vivo absorption microspectroscopy of both the thylakoid

compartments and the eyespot. The decomposition of the overall absorption spectra in pigment constituents indicates that Cr6+ induced

a complete pheophinitization of the chrorophylls and a modification of the carotenoids present in the eyespot only when its

concentration is equal or greater than 10mM. Due to this low tolerance level, C. reinhardtii could be used as indicator of Cr pollution, but

it is not feasible for bioremediation purposes.

r 2007 Elsevier Inc. All rights reserved.

Keywords: Heavy metal; Chromium; Photosynthesis; Algal pigments

1. Introduction

Algae are the basis of the food chain in all aquaticecosystems. The nutritional value of an alga depends upondiverse characteristics including shape, size, composition,digestibility, and toxicity. However, it is the biochemicalcomposition (fatty acids, sterols, amino acids, sugars,minerals, and vitamins) that acts as a determinant inestablishing the nutritional quality transferred to the othertrophic levels of the food chain (Brown and Miller, 1992).Clearly, stress environmental conditions that affect thebiochemical composition of the alga will have a majorimpact on its food value.

Heavy metals such as copper and zinc can be essentialtrace elements for photosynthetic organisms. In higher

e front matter r 2007 Elsevier Inc. All rights reserved.

vres.2007.01.011

ing author. Fax: +39 050 315 2760.

ess: paolo.gualtieri@pi.ibf.cnr.it (P. Gualtieri).

concentrations, however, these metals as well as other non-essential metals such as chromium, lead, mercury andcadmium, have severe toxic effects. Heavy metals, ulti-mately derived from a growing number of diverse anthro-pogenic sources (industrial effluents and wastes, urbanrunoff, sewage treatment plants, boating activities, agri-cultural fungicide runoff, domestic garbage dumps, andmining operations) have progressively affected an increas-ing number of varied ecosystems (Sinha et al., 2002).Toxicity may result in diverse effects, which depend on

the type of algae, the nature and concentration of themetal, and the environmental conditions accompanyingheavy metal stress (Satoh et al., 2005). Interestingly,nutrient availability (in particular, nitrogen) dramaticallyincreases the ability of algae to accumulate heavy metals,suggesting that agricultural runoff into fresh water andcoastal areas will greatly increase the entry and theconsequent concentration of heavy metals into the food

ARTICLE IN PRESSM.C. Rodrıguez et al. / Environmental Research 105 (2007) 234–239 235

chain (Wang and Dei, 2001). This concentration may occurthrough the action of chelators, which directly bind andstore the metal ions, as well as by induction of defensemechanisms that allow the cell to reduce the toxic effects ofthe metals.

Generally, heavy metal toxicity is related to the oxidativedamage induced in living systems, which can be promotedboth by directly increasing the cellular concentration ofreactive oxygen species (ROS) and by reducing the cellularantioxidant capacity (Livingstone, 2001). ROS can beextremely harmful to organisms at high concentrations.They can oxidize proteins, lipids, and nucleic acids, oftenleading to alterations in cell structure and mutagenesis. Toadd to the problem, chloroplasts have a complex system ofmembranes rich in polyunsaturated fatty acids, which arepotential targets for peroxidation. Therefore, modulationof chloroplastic antioxidants seems to be a particularlyimportant strategy, allowing the algae to acclimate to theenvironmental stress (Okamoto et al., 2001). Under acuteconditions, however, the toxic effects of the pollutants mayoverwhelm the antioxidant defenses. This may result in celldeath or the shut down of all cellular machinery. Growthinhibition and chlorosis are common symptoms of metalphytotoxicity in several algae, in which photosynthesis isprobably the most affected metabolic process (Ali et al.,2006).

Chromium (Cr), which is not essential for any metabolicprocess, is the seventh most abundant metal in the earth’scrust (Katz and Salem, 1994). It occurs in nature aschromite, a compound formed with oxygen and iron. Crconcentrations in non-polluted waters vary from 1 to10 mM in fresh waters and from 0.03 to 1 mM in oceanicwaters, but levels as high as 1.5 and 48mM have beenreported for paper mill effluents and tanning wastewaters,respectively (Cervantes et al., 2001).

In natural environments, Cr occurs in several oxidationstates, ranging from Cr2+ to Cr6+, with trivalent andhexavalent states being the most stable and frequent in theterrestrial environment. Both Cr3+ and Cr6+ differ interms of mobility, bioavailability and toxicity (Panda andParta, 1997). Cr3+ is routinely used for leather tanningbecause it forms stable complexes with amino groups inorganic material. In presence of excessive oxygen, Cr3+

oxidizes into Cr6+, which is highly toxic and more solublein water than other forms. Cr6+ can easily cross themembrane, whereas the phosphate–sulphate carriers alsotransport the chromite anions, Cr2O4

2�. On the other hand,Cr3+ does not utilize any specific membrane carrier andhence enters into the cell through simple diffusion. Thediffusion is possible only after the formation of appropiatelipophilic ligands (Panda and Choudhury, 2005a).

Depending on pH, Cr6+ forms hydrochromate(HCrO4

�), chromate (CrO42�) and dichromate (Cr2O7

2�)and is highly soluble in water. At pH values below 6.2, thehydrochromate anion is predominant while at pH above7.8, the chromate ion dominates. Cr3+ on the other hand isless soluble in water and is required in trace amounts as an

inorganic nutrient for animals. Cr phytotoxicity can resultin degradation of pigment status, nutrient balance,enhancement of antioxidant enzymes activity, and induc-tion of oxidative stress in plants and algae (Wong andChang, 1991; Rai et al., 1992; Cervantes et al., 2001; Pandaand Choudhury, 2005b), together with alteration ofchloroplast and membrane ultrastructure (Bassi et al.,1990; Choudhury and Panda, 2005a). Cr, mostly in itshexavalent form, has been shown to generate OHd radicalsfrom H2O2, to replace Mg ion from the active site of manyenzymes (Vajpayee et al., 2000) and to induce both thedegradation of carotenoids (Rai et al., 1992), and theincrease of carotenoids. This increase may act as anantioxidant mechanism to scavenge ROS generated inresponse to Cr toxicity (Rai et al., 2004).In the present study we analyzed the effects of Cr in the

thylakoid compartments and eyespot of the unicellular algaChlamydomonas by measuring the pigment compositionevolution after the addition of this heavy metal in vivo bymicrospectroscopy. The decomposition of the absorptionspectra in pigment components showed that Cr atconcentration higher than 10 mM causes the completepheophinitization of both chlorophyll a and b. Thisevolution is associated with the inhibition of the enzymesinvolved in the cyclization process of the b-ionone ring.

2. Materials and methods

2.1. Cultures

Wild-type Chlamydomonas reinhardtii was kindly provided by Drs. K.

Niyogi and R. Dent, Department of Molecular and Plant Biology,

University of California at Berkeley, USA. Cells were grown in TAP

medium supplemented with sodium acetate at pH 6.8 (Harris, 1989) at

constant temperature of 24 1C and under continuous agitation and

illumination (2� 102 mmol photonsm�2 s�2). Cr was added to the basal

medium as K2Cr2O7 to the required final concentrations, i.e. 1, 5, and

10mM. Modelling initial culture media conditions in software MINEQL+

4.0 indicated that the totality of Cr was in solution as dichromate anion

for all the assayed concentrations (Environmental Research Software,

Inc.) Measurements were performed after 48 h of incubation with Cr.

2.2. Microscopy

Cells harvested by low-speed centrifugation (1000g, 5min) were

examined with a Zeiss Axioplan microscope (Zeiss, Germany) equipped

with a 100� (N.A. 1.3) planapochromatic objective, and a 100W mercury

lamp.

2.3. Photography

Photographs were recorded with an Olympus Camedia C-30303 digital

camera (Olympus, Japan) mounted on the Zeiss Axioplan microscope

(Zeiss, Germany).

2.4. Photoaccumulation experiments

To verify the presence of photoaccumulation behavior, Chlamydomo-

nas cells were placed in a Petri dish completely covered with black self-

sticking paper except for a 2 cm diameter hole on the top of the dish. The

populations were screened for photoaccumulation in liquid medium. A cell

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Fig. 1. (a) Untreated Chlamydomonas reinhardtii cell. The visible orange

spot on the left of the cell is the eyespot. The two flagella are visible in the

upper part of the figure. Bar: 8 mm. (b) 10mMCr treated C. reinhardtii cell.

The visible orange spot in the center of the cell is the eyespot. The arrow

points to the carbohydrate granule. Bar: 8 mm.

Fig. 2. Absorption spectra of the thylakoid membrane system of an

untreated Chlamydomonas reinhardtii cell (black line). Main pigments

(gray and discontinuous lines) are shown together with their maxima.

M.C. Rodrıguez et al. / Environmental Research 105 (2007) 234–239236

concentration of 5� 106 cellsmL�1 was used for both populations. The

dishes were illuminated from above with an incandescent lamp

(2� 102 mmol photonsm�2 s�1). Photographs were taken after 1 h expo-

sure.

2.5. Velocity determination

A Pulnix TM860 (Pulnix, USA) CCD video camera was mounted onto

a Zeiss Axioplan microscope (Zeiss, Germany) equipped with 16� and

60� objectives and 100W halogen lamp as light source. Cells were placed

in a small chamber obtained by fixing a PVC ring onto a microscope slide.

The chamber was closed by means of a cover slip so as to avoid sample

drying-out. The signal of the camera was the input of a Scion LG-3 Frame

Grabber (Scion Corporation, USA) plugged into a Pentium III personal

computer 750MHz clock. For the automatically velocity determination

experiment, a sequence of images taken at increasing intervals of time,

stored, and processed using the method of Coltelli et al. (2006).

2.6. Absorption microscopy

Absorption spectra in the visible range were measured in vivo on

photosynthetic compartments (thylakoid membranes of chloroplasts) and

on the eyespot of single cells. The apparatus used to perform in vivo

measurements was homemade and described in detail in Gualtieri et al.

(1989).

All the absorption spectra were recorded from 400 to 700 nm, with a

step size of 0.5 nm and scan rate of 100 nm s�1. For each wavelength

10,000 values of optical density were averaged.

2.7. Mathematical computation of absorption spectra

The relative contribution of the individual pigment classes to

absorption spectra can be estimated by curve-fitting routine using

templates of the absorption spectra of the pigments present in the alga

(Barsanti et al., 2007), namely chlorophylls, pheophytines, and carote-

noids.

The generation of templates of pigment absorption spectra is based on

the representation of the spectral dependence of the pigment absorption in

terms of a small number of asymmetric Gaussian bands. These templates

can be then combined together to obtain the spectrum of each

photosynthetic compartment. The quantitative contribution of each

pigment is achieved by measuring the surface area of the relative Gaussian

curve. For details on spectral decomposition refer to Evangelista et al.

(2006).

3. Results

To analyze the effects of Cr on the photosyntheticmetabolism of Chlamydomonas three increasing concentra-tions of the metal were tested: 1, 5, and 10 mM.

After 48 h of incubation in the presence of 1 and 5 mM,cell morphology appeared unchanged with respect to theuntreated cells (Fig. 1a). Photoaccumulation behavior waspresent; velocity of the cells treated with 1 mM Cr was197 mms�1, that of the cells treated with 5 mM was198 mms�1, and both were comparable to that of thecontrol, 202 mms�1. The chloroplast retained its greenpigmentation, and the eyespot appeared bright orange.Since no difference in the spectral features could bedetected between the treated and untreated cells, Fig. 2shows the absorption spectrum measured on the chlor-oplast of an untreated cell. Its absorbance is 0.8, and thepositions of the major peaks of the decomposed pigments

are indicated on the spectrum. The main maxima ofchlorophyll a are 432.5, 582.5, 628.5, and 678 nm; those ofchlorophyll b are 449, 597, 620.5, and 651 nm, whereascarotenoids show the main maximum at 483 nm.When incubated with 10 mM Cr, cells became completely

discolored; photoaccumulation behavior was completelyabsent since cells were immotile. Microscopy observationshowed alteration of the chloroplast matrix and morphol-ogy, and a darkening of the eyespot hue, which appeareddeep red-orange (Fig. 1b). Moreover, the cells containedlarge starch granules (arrow, Fig. 1b). Absorption spec-trum of the chloroplast reflected the changes of the cells(Fig. 3): absorbance showed a dramatic decrease in thetreated cells (A ¼ 0.07). Spectral decomposition shows thatchlorophyll a and b converted to pheophytin a and b, andthe carotenoids curve revealed hypsochromic and bath-ochromic bands not present in the spectrum of theuntreated cells. The positions of the major peaks of the

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Fig. 3. Absorption spectra of the thylakoid membrane system of a 10mMCr treated Chlamydomonas reinhardtii cell (black line). Main pigments

(gray and discontinuous lines) are shown together with their maxima.

Fig. 4. Absorption spectra of the eyespot of an untreated Chlamydomonas

reinhardtii cell (dark line) and of a 10 mM Cr treated Chlamydomonas

reinhardtii cell (gray line).

M.C. Rodrıguez et al. / Environmental Research 105 (2007) 234–239 237

decomposed pigments are indicated on the spectrum. Themain maxima of pheophytin a are 417, 512, 540, 614, and668 nm; those of pheophytin b are 432, 522.5, 549.5, 600.5,and 652.5 nm, whereas carotenoids show two main maximaat 454.5 and 574 nm.

Fig. 4 show the absorption spectra recorded on theeyespot of a cell after 48 h incubation with 10 mM Cr(continuous line) and the eyespot of an untreated cell(dotted line). The absorption spectra recorded on theeyespot of the cells treated with 1 and 5 mM Cr are notshown, since these lower Cr concentration do not producesignificant effects on the absorption characteristics of theorganelle. In the case of 10 mM Cr, the absorbance was0.10, while it was 0.6 in untreated cells; the absorptionmaximum underwent a shift from 451 to 478 nm due to thepresence of carotenoids that absorb at longer wavelengths.

In untreated cells the ratio between Chlorophyll a and b

content calculated as ratio of the surface area of thecorresponding decomposition curves is 2.24. In 10 mMtreated cells the ratio between pheophytin a and b

calculated in the same way was 2.17. The amount ofcarotenoids as percent amount of chlorophylls (untreatedcells) or pheophytins (10 mM treated cells) changed from0.36 to 0.94.

4. Discussion

Heavy metals disturb the oxidative balance in algae, andthus an important palliative measure is the induction ofantioxidants mechanisms (Pinto et al., 2003); thereforealgae have developed a wide range of protective mechan-isms that serve to remove ROS before they can damagesensitive parts of the cellular machinery (Clijsters et al.,1999). These can be conveniently divided into lowmolecular weight compounds, such as carotenoids, ortocopherols and ascorbic acid, as well as enzymaticcatalysts of high molecular weight, such as superoxidedimutase, ascorbate peroxidase or catalase (Okamoto etal., 1996). Carotenoids such as fucoxantin, peridinin,astaxanthin, and b-carotene are the most abundantcarotenoids found in the aquatic environment and arelocated principally in the chloroplasts. The role of thesecarotenoids is two-fold: not only do they aid in broadeningthe spectrum of PAR but they also protect the light-harvesting pigments in the antenna complexes againstphotochemical damage caused by excited triplet states andother ROS by acting as quenchers (Okamoto et al., 2001).In general, quenching efficiency is directly proportional tothe number of conjugated double bonds.Though many ROS-generating processes are slow under

normal conditions, heavy metals together with environ-mental factors such as high irradiation or UV exposure canaccelerate them. Higher levels of antioxidants would becritical to withstand photo-oxidative stress elicited by areduced energy-utilizing capacity, as a consequence ofmetal toxicity (Kupper et al., 2002).The induction of antioxidants in response to enhanced

ROS production is generally proportional to the durationand severity of the stress applied to algal cultures, beingdifferent in the case of chronic or acute treatment withheavy metals. Enhanced levels of cellular antioxidantscould allow the cells to achieve acclimation to increasedsteady-state concentrations of ROS during chronic stress.In contrast, the abrupt generation of high levels of ROSover a short period of acute stress will usually exceed thetotal antioxidant capacity (Kupper et al., 2002).Our results showed that Chlamydomonas cells treated

with 1 and 5 mM Cr can modulate chloroplastic antiox-idants and acclimate to the oxidative stress induced by theheavy metal, attenuating or eliminating the damage to thephotosynthetic machinery. Cell morphology is not affected,as well as chloroplast and eyespot pigmentation (Figs. 1a,2, and 4), which are typical of Chlorophyceae (Barsanti

ARTICLE IN PRESSM.C. Rodrıguez et al. / Environmental Research 105 (2007) 234–239238

and Gualtieri, 2006); moreover, motility is retained andphotoaccumulation behavior is normally present. Whenthe concentration of Cr increases to 10 mM, the exposure tothe metal becomes acute; under these conditions, the levelsof ROS formed exceed the ability of the antioxidant systemto cope with them, and damage to cellular compoundsoccurs. The absorbance of the photosynthetic compart-ments undergoes a dramatic decrease from 0.8 to 0.07, andpheophytins replace chlorophylls. Thylakoid lipids areespecially susceptible to oxidative damage because of theabundance of unsaturated fatty acid chains. Reactiveoxygen attack of these lipids initiates peroxyl-radical chainreactions, which eventually can destroy the thylakoidmembrane, by indirect damage of structural pigment–pro-tein complexes located in the thylakoid membrane.Residual patches of membranes still hosting active photo-synthetic centers, will undergo pheophytinization of thechlorophylls due to exposition to acidic content of thethylakoid lumen and the consequence substitution ofMg2+ by the hydrogenions; this would account for theabsorbance decrease in the treated cells even if pheopythinshave higher extinction molar coefficient in the Soret band(Eijckelhoff and Dekker, 1997) (Fig. 3), and for the almostidentical ratio between chlorophylls (2.24) and pheophytins(2.17).

The stress treatment induced an increased synthesis ofantioxidant compounds within subcellular sites highlyprone to oxidative stress such as chloroplasts. Therefore,the ratio of carotenoids to pheophytins in 10 mM treatedcells is much higher (0.94) than the ratio of carotenoids tochlorophylls in untreated cell (0.36), though the absoluteamount is lower. At this concentration, the cells becomeimmotile, and are not capable of photoaccumulation,indicating an irreversible damage of the metabolic machin-ery.

Another stress feature of the treated cells is the shift ofthe absorption maximum of the eyespot towards longerwavelengths, from 451 nm in untreated cells to 478 nm intreated cells, with a great decrement of absorbance (Fig. 4).This could indicate a predominance of acyclic carotenoidssuch as lycopene with respect to carotenoids containing ab-ionone ring, such as b-carotene. This selective accumula-tion could be due to the destruction of membrane sitescontaining enzymes necessary to the cyclization of lycopeneto cyclic carotenoids.

Also the carbohydrate accumulation visible in treatedcells (arrow, Fig. 1b) indicates the impact of Cr on cellmetabolism. The decrease in the rate of utilization of thephotosynthate and the consequent accumulation could belikely a consequence of an altered source–sink relationship,mainly due to the inhibition of photosynthesis and celldivision processes (Brent Nichols et al., 2000).

These data indicate that Chlamydomonas is very sensitiveto heavy metal stress even and at very low concentrations.For example Chlorella pirenoidosa (Horcsik et al., 2006),Dunaliella salina (Donmez and Aksu, 2002), Euglena

gracilis (Rocchetta et al., 2006) can tolerate much higher

value. Therefore, Chlamydomonas reinardhii can be con-sidered feasible for detecting Cr pollution in waterresources, but too sensitive to act as an effectivebioremediation organism.

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