Cadmium exerts its toxic effects on photosynthesis via acascade mechanism in the cyanobacterium, SynechocystisPCC 6803pce_2537 2075..2086

TÜNDE TÓTH, OTTÓ ZSIROS, MIHÁLY KIS, GYOZO GARAB & LÁSZLÓ KOVÁCS

Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, PO Box 521, H-6726 Szeged,Hungary

ABSTRACT

Despite intense research, the mechanism of Cd2+ toxicity onphotosynthesis is still elusive because of the multiplicity ofthe inhibitory effects and different barriers in plants. Thequick Cd2+ uptake in Synechocystis PCC 6803 permits thedirect interaction of cadmium with the photosyntheticmachinery and allows the distinction between primary andsecondary effects. We show that the CO2-dependent elec-tron transport is rapidly inhibited upon exposing the cells to40 mM Cd2+ (50% inhibition in ~15 min). However, duringthis time we observe only symptoms of photosystem Iacceptor side limitation and a build of an excitation pres-sure on the reaction centres, as indicated by light-inducedP700 redox transients, O2 polarography and changes inchlorophyll a fluorescence parameters. Inhibitory effects onphotosystem II electron transport and the degradation ofthe reaction centre protein D1 can only be observed afterseveral hours, and only in the light, as revealed by chloro-phyll a fluorescence transients, thermoluminescence andimmunoblotting. Despite the marked differences in themanifestations of these short- and long-term effects, theyexhibit virtually the same Cd2+ concentration dependence.These data strongly suggest a cascade mechanism of thetoxic effect, with a primary effect in the dark reactions.

Key-words: cadmium; carbon metabolism; cyanobacteria;excitation pressure; photosynthesis.

Abbreviations: CA, carbonic anhydrase; CCM, carbon-concentrating mechanism; ET, electron transport; FNR,ferredoxin/NADP+-oxidoreductase; MV, methyl viologen;pBQ, p-benzoquinone; ROS, reactive oxygen species;Rubisco, ribulose bisphosphate carboxylase-oxygenase; TL,thermoluminescence; WOC, water oxidizing complex.

INTRODUCTION

Cadmium is one of the most toxic environmental pollutantsand several studies focus on the mechanism of action of this

element. For a long time, cadmium was regarded as a toxicheavy metal without any biological function. Some recentdiscoveries revealed that plants can require Cd2+ at traceamounts for optimal growth (Liu et al. 2008). Althoughcadmium is a relatively rare element in the Earth crust(Shiller & Boyle 1987), human activities can increase itsconcentration to toxic level. Cadmium is introduced intothe terrestrial environment from mining, non-ferrousmetal production, phosphate fertilizers and manure thatcan increase the Cd2+ concentration up to a few hundredmg kg-1 soil at industrialized or intensely cultivated areas.In addition, cadmium input to the aquatic environment isthrough discharge of industrial waste, surface run-off anddeposition. This elevated Cd2+ concentration can result inthe uptake and accumulation of Cd2+ in different organismsto toxic levels. Cd2+ exerts severe physiological effects inmost living organisms also because it has a relatively longbiological half-time, for example, in human it is estimatedto be between 25 and 50 years depending on age (Wagner1993). Because of this long half-time, plants and animalscan easily accumulate Cd2+ in their organs. Plants and phy-toplankton possess high Cd2+ accumulating capacities(Conway 1978). As the Cd2+ that is incorporated into plantsor aquatic cells is transferred to higher trophic levels of thefood chain, consecutive accumulation at every trophic level,the so-called biomagnifications, can increase the Cd2+ con-centration in the consumers much above the Cd2+ concen-tration found in the environment.

The photosynthetic apparati of various photoautotrophorganisms appear to be particularly susceptible to thisheavy metal pollutant. In spite of the intense research onCd2+ effects on photosynthesis, the mechanism of Cd2+ tox-icity is still elusive and the reported results are often con-tradictory.The toxic effect of Cd2+ on photosynthesis is verycomplex and apparently different mechanisms are involved.Cd2+ can interact with proteins by reacting with their activegroups (Clijsters & Vanassche 1985; Vanassche & Clijsters1990) or replacing metal cations (Zn2+, Cu2+, Ca2+) in met-alloproteins (Vanassche & Clijsters 1990; Grzyb et al. 2004;Faller, Kienzler & Krieger-Liszkay 2005). Heavy metalscan generate reactive oxygen species (ROS) by Fenton’sreaction although in this respect Cd2+ is rather inert.Many proteins involved in photosynthetic and related

Correspondence: L. Kovács. Fax: +3662433434; e-mail: kovacs.laszlo@brc.mta.hu

Plant, Cell and Environment (2012) 35, 2075–2086 doi: 10.1111/j.1365-3040.2012.02537.x

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processes contain Zn2+ [e.g. carbonic anhydrases (CAs)]and sulfhydryl groups (e.g. glyceraldehyde-3-phosphatedehydrogenase, ribulose-5-phosphate kinase, fructose-1,6-bisphosphatase), which may make photosynthesis one ofthe main targets to Cd2+.

The primary target of Cd2+ inhibition is still not identifiedand it is not clear which are the direct and indirect effects.Both the donor (Sigfridsson et al. 2004; Faller et al. 2005)and the acceptor sides of photosystem II (PSII) (Atal,Saradhi & Mohanty 1991; Sigfridsson et al. 2004) have beendemonstrated to suffer from damage upon Cd2+ exposure invitro, but these results have not been confirmed by in vivoexperiments. Recently, photosystem I (PSI) acceptor sideand ferredoxin-NADP reductase (FNR) were also sug-gested as the primary target (Grzyb et al. 2004), but in theseexperiments the authors used unrealistically high millimo-lar to molar Cd2+ concentrations. In other studies, the effectof Cd2+ on the dark reactions of photosynthesis was empha-sized (Krupa, Oquist & Huner 1993; Aravind & Prasad2004). The contradictory observations may originate fromdifferences in the experimental conditions, particularly inthe applied Cd2+ concentration, the duration of Cd2+ expo-sure and the way the Cd2+ was administered. Furthermore,the mechanism of Cd2+ toxicity can be different in variousorganisms. In case of terrestrial plants, cadmium is typicallytaken up from the soil by the root system, from where it istransported to the assimilatory tissues of leaves and canreach the chloroplasts. The transport of cadmium from thesoil to the chloroplast via different tissues is delayed byseveral barriers. Active defensive mechanisms such as theexpression of metal sequestering proteins and the accumu-lation of Cd2+ in the vacuole sequester a considerableamount of Cd2+ away from potential targets (Cobbett &Goldsbrough 2002).To affect photosynthesis, the remainingCd2+ must be transported to the leaves from the root viaxylem transport. By the time the Cd2+ could accumulatesignificantly in the chloroplast, secondary effects induced inthe roots can already influence the photosynthetic machin-ery, for example, the iron deficiency caused by the inhibitionof the metal transport leading to a disturbed metal homeo-stasis that can suppress the chlorophyll and metalloproteinsynthesis (Siedlecka & Baszynski 1993).

In contrast to terrestrial plants, aquatic organisms canreadily take up Cd2+ from water. Cd2+ has high water solu-bility compared with other transition metals, which ensureshigh mobility to Cd2+ in marine and fresh water environ-ments and consequently promotes its wide distribution inaquatic ecosystems. The species of phytoplankton andhigher plants submerged to water take up metals via thewhole surface of their body. They possess very narrowcuticle and thin cell wall and thus there is no need for longtransport mechanisms between different tissues and organs.Cd2+ has a quick access to the chloroplast or, in cyanobac-teria, the thylakoid membranes, and can rapidly exerteffects on photosynthesis. Hence, while in terrestrial plantsthe secondary effects of Cd2+ can dominate above the directeffects on photosynthesis, in aquatic photosynthetic organ-isms direct effects can play the major role, at least in the

first phase of Cd2+ inhibition. The cadmium-induced effectscomparable to responses observed in cyanobacteria can beobtained in higher plants only in isolated protoplasts orchloroplasts (Greger & Ogren 1991).

Cyanobacteria are important part of phytoplanktonthat support all aquatic ecosystems and are known to behighly sensitive to cadmium. Cyanobacteria possess highCd2+-binding capacity and rapid uptake, which make theseorganisms employable in phytoremediation to eliminateCd2+ from polluted areas (Rangsayatorn et al. 2002). Theenhanced Cd2+ tolerance is a prerequisite for such kind ofbiotechnological application and requires a detailed under-standing of the inhibitory mechanism of Cd2+.To this end, inthe present study, we investigated direct and indirect effectsof Cd2+ on photosynthesis of the cyanobacterium Syn-echocystis PCC 6803.

MATERIALS AND METHODS

Organism and culture conditions

Synechocystis PCC 6803 cells were grown photo-autotrophically in BG11 medium supplemented with20 mm N-(2-hydroxyethyl)piperazine-N′-ethanesulfonicacid (HEPES)-NaOH (pH 7.5), at 30 °C under continuousillumination at 25 mmol photons m-2 s-1 photon flux density.Cultures were aerated on a horizontal shaker at 100 r.p.m.The Chl concentration was determined according to Arnonet al. (1974). Different concentrations of CdCl2 (5, 10, 20, 40,100, 200 mm) were applied during the exponential phase ofthe cell culture.

Measurement of photosynthetic activities

Photosynthetic oxygen-evolving activity of intact cells wasmeasured with a Clark-type oxygen electrode (HansatechInstruments, Kings Lynn, UK) at saturating intensity of2000 mmol photons m-2 s-1 white light at 30 °C. The PSII-dependent Hill reaction was recorded in the presence of1 mm para-benzoquinone (pBQ).The reaction from H2O toPSI was followed in the presence of 1 mm methyl viologen(MV). For all polarography measurements, the sameamount of sample equivalent to 2 mg mL-1 chlorophyll wasloaded into the sample holder.

Measurement of chlorophyll a fluorescencetransients

Chlorophyll a fluorescence transient emission was detectedwith a Handy PEA instrument (Hansatech Instruments,King’s Lynn, Norfolk, UK). For the measurements, thesample (equivalent to 20 mg chlorophyll) was filtered onto afilter paper disk. Synechocystis cells were dark adapted for3 min before the measurements then the cells were illumi-nated with continuous light (650 nm peak wavelength,300 mmol photons m-2 s-1 maximum photon flux density)provided by an array of three light-emitting diodes (LEDs)focused on a circle of 5 mm diameter of the sample surface.

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The first reliably measured point of the fluorescence tran-sient is at 20 ms, which was taken as Fo.

Maximum and effective quantum yield measurementsof PSII were carried out by using a Pulse-Amplitude-Modulation chlorophyll fluorometer (PAM-101); 1 cmoptical pathlength was used and the chlorophyll concentra-tion of the sample was 2 mg mL-1.The actinic light was givenby 102 L (Walz, Effeltrich, Germany) red LED light sourceswith 650 nm peak wavelength, 40 mmol photon m-2 s-1

maximum photon flux density. To measure the maximalfluorescence 1 s long, 3000 mmol photons m-2 s-1 maximumphoton flux density pulse was used. During the illumina-tion, the maximal fluorescence (Fm′) was measured every200 s.

Measurement of the oxidation–reductionkinetics of P700

The light-induced redox changes of P700 were monitoredby measuring the absorbance changes at 820 nm with aDual PAM-100 chlorophyll fluorometer (Heinz Walz). Thesample (equivalent to 20 mg chlorophyll) was filteredonto a filter paper disk and cells were dark adapted for3 min before the measurements. In order to determinethe amount of total photo-oxidizable P700, 10 s far-redpre-illumination followed by a 200 ms long pulse with1000 mmol photons m-2 s-1 maximum photon flux densitywas used. The rate of the cyclic electron transport (ET) wascharacterized by the time constants (t) of the re-reductionof P700+ after pre-illumination. The decay kinetics wasrecorded with 5 ms time resolution and fitted with a singleexponential.

Thermoluminescence measurements

Thermoluminescence (TL) measurements were carried outin a home-built apparatus (Demeter et al. 1984). For themeasurement, the sample (equivalent to 20 mg chlorophyll)was filtered onto a filter paper disk. After 3 min dark adap-tation at 30 °C in the sample holder, the cells were excitedat -20 °C by a saturating single-turnover flash. Immediatelyafter excitation, the sample was cooled down to -30 °Cand the emitted TL was measured during heating of thesample in the dark at a heating rate of 20 °C min-1. The QTL band was detected in the presence of 10 mm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU).

Polypeptide analysis

For detailed measurements of D1 content after the differ-ent treatments, cells with the same chlorophyll content werehomogenized in 250 mL 2¥ Laemmli buffer, and the homo-genates were incubated at 90 °C for 5 min followed by a20 min long incubation at 37 °C. D1 protein of the wholecell extract was separated and analysed by immunoblottingas described by (Bajkan et al. 2010).

RESULTS

Toxic effect of Cd2+ on the cyanobacterium SynechocystysPCC 6803 is demonstrated by measuring the growth rate ofalgal cultures exposed to various CdCl2 concentrations inthe range between 5 and 100 mm (Fig. 1).The increase of celldensity and chlorophyll concentration was followed for 3 d.Even at the lowest applied concentration, at 5 mm, Cd2+

significantly suppressed the growth rate (Fig. 1a); above

Figure 1. Variations in the cell density, measured as the optical density (OD) at 750 nm (a), and the chlorophyll content (b) inSynechocystis culture as a function of time after the Cd2+ treatment at different CdCl2 concentrations, as indicated.

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10 mm, the cell propagation was completely halted. Thechlorophyll content of the cultures showed similar changes(Fig. 1b). In the presence of 5 mm Cd2+, the increase of chlo-rophyll concentration was retarded to the same extent asthe increase of cell density; at higher concentrations, theydid not increase at all. It is interesting to note that despitethe complete inhibition of cell propagation at 10 mm Cd2+,we did not observe any decrease in the cell density andpigment content even at four times higher concentration ofCd2+ during the 3 d exposition of the cells to Cd2+. Further,the ratio of chlorophyll content to cell density remainedunchanged at all examined concentrations. These datastrongly suggest that although the cells are not able todivide they still keep their viability at least for 3 d in thepresence of Cd2+ in the solution. This conclusion is furthersupported by viability test (Supporting Information Fig.S1), which has shown that the cells keep their divisionability similar to the untreated control even after 12 h Cd2+

treatment if the cells are subsequently washed with Cd2+-free culture medium and transferred to agar plates contain-ing BG11 medium.

The high Cd2+ sensitivity of the photosynthetic ET inSynechocystis cells in vivo is demonstrated in Fig. 2, datasimilar to our previous work (Sas et al. 2006). Cd2+ at 40 mmconcentration brings about a complete block in the whole-chain ET in about half an hour. In order to localize theprimary site of inhibition in the photosynthetic ET, weinvestigated the effect of cadmium on the photosyntheticactivity of intact cells in the presence of artificial electron

acceptors, pBQ and MV (Fig. 2). It can be seen, however,that neither the pBQ- nor the MV-dependent Hill reactionrates showed significant decrease even 60 min after the Cd2+

treatment; by this time, the whole-chain ET had beenalready completely blocked. In the presence of pBQ andMV, respectively, the PSII-dependent oxygen evolution andthe PSII + PSI ET activity, which are independent of ferre-doxin and the reduction of NADP+, can be assessed. Hence,these data show that the site of inhibition is to be found ator beyond the ferredoxin-binding site in PSI.

The oxidation-reduction kinetics of PSI reaction centre,P700, provides useful information about PSI functions invivo. The polyphasic oxidation kinetics of P700 upon illu-mination by actinic red light (Fig. 3) can be detected incontrol sample by measuring the absorption change at820 nm (Harbinson & Hedley 1993). The first, fast phase ofoxidation is related to the reduction of ferredoxin. It isfollowed by a temporary reduction and, finally, a fewminutes long oxidation occurs in the last phase, which isattributed to the Calvin–Benson cycle. In the sampletreated with 40 mm CdCl2, the initial fast phase is still

Figure 2. Effect of 40 mm CdCl2 on the rate of electrontransport in the absence and presence of artificial electronacceptors (1 mm pBQ or 1 mm MV) in Synechocystis cells as afunction of time after the Cd2+ treatment in the presence of thegrowth light (25 mmol photons m-2 s-1 white light). The data aregiven in percentage of the corresponding control values. Theactivities corresponding to 100% for the untreated cells areabout 200, 150 and 350 mmol O2 mg-1 (chl) h-1, for whole-chainelectron transport, MV and pBQ-dependent Hill reactions,respectively. Values are given as means � SD (n = 3).

Figure 3. Kinetics of P700 oxidation recorded as absorbancechanges at 820 nm in control (a) (60 min dark adapted) andCd2+-treated (b) (40 mm CdCl2, 60 min dark) cells. Before the5 min long actinic light (AL: 50 mmol photons m-2 s-1) Pm, and atits end Pm′ were obtained with a saturation pulse (SP). Arrowsindicate the onset of AL and SPs.

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present after 1 h incubation, but the slow kinetic compo-nent is missing, suggesting the suppressed capacity of theCalvin–Benson cycle.

By monitoring the reduction kinetics of P700 after far-red(FR) illumination, the PSI cyclic ET can be tested, which isrelated to the PSI acceptor side function; because PSI ispreferentially excited by FR light, the contribution of PSIIto the re-reduction of P700+ is negligible (Bernat et al.2011).After the illumination of the cells with FR light, a fastre-reduction occurs at a rate proportional to the rate ofcyclic ET, and can be characterized by the time constantof the dark reduction. It can be seen that comparabledecay time was observed in dark-adapted control and40 mm CdCl2-treated samples (Table 1). After 5 min pre-illumination by actinic light, the cyclic ET in both samples isenhanced as the result of accumulation of NADPH duringillumination. In the presence of Cd2+, pre-illuminationproved to be more effective in enhancing the re-reductionrate of P700+ compared with the control, which suggests anincreased NADPH/NADP ratio in Cd2+-treated samples.

In order to further clarify the site of inhibition in thewhole-chain ET, that is the site of inhibition of O2 evolutionin the absence of artificial electron acceptors, we also per-formed chlorophyll fluorescence analysis (Fig. 4). Duringillumination with non-saturating light intensity, the electronacceptors of PSII become partly reduced through acomplex kinetics and finally reach a steady-state level withfluorescence intensity (Fs), a value between the maximalfluorescence (Fm) and the original fluorescence (Fo) levels.The Fs level depends on the applied actinic light intensity.The untreated control and the cells pre-incubated with40 mm Cd2+ for 1 h in the dark exhibit the same Fv/Fm valuesof about 0.4, which is characteristic to dark-adapted Syn-echocystis cells (Campbell et al. 1998; Laczko-Dobos et al.2008; Hackenberg et al. 2009). In light-adapted state, themaximal fluorescence, Fm′, and the Fs parameters can beused to determine the effective quantum yield of PSII byusing the FPSII = 1 - Fs/Fm′ formula (Baker, Harbinson &Kramer 2007). It can be seen that after illumination of thesamples at a photon flux density of 40 mmol photons m-2 s-1

actinic red light, significantly lower FPSII can be obtainedfrom Cd2+-treated sample than in the control. The decrease

of FPSII upon Cd2+ treatment is mainly due to the elevationof Fs value while the decrease of Fm′ is negligible. The addi-tion of 1 mm MV restores Fs to the control level, whichindicates a PSI acceptor side limitation.

In order to identify the possible long-term effects of Cd2+

on the photosystems, functional parameters of PSI and PSIIwere followed for several hours. In longer timescale, theFv/Fm and the amount of the maximum photo-oxidizableP700 (Pm) parameters were monitored. The activity of PSI(Pm) did not show significant changes during the studied timeinterval; in contrast, a continuous decrease in PSII maximalquantum yield can be observed (Fig. 5). The tendency ofFv/Fm (around 0.4 in control cells) to decrease became pro-nounced after 4 h of the Cd2+ treatment, and in approxi-mately 12 h, a very low value (around 0.15) was detected.

The effect of Cd2+ on PSII was further examined by TL.TL is a suitable technique to examine the charge separationand stabilization in PSII (Ducruet & Vass 2009). In cyano-bacterial cells, TL signal can be induced by a saturatingsingle turnover flash at -20 °C. The arising B band, with a

Table 1. The effect of cadmium on the time constant (t) ofre-reduction of P700+ kinetics

t (ms � SD)

Dark adapted Light adapted

Control 280 � 21 143 � 240 mm CdCl2 treated 261 � 9 59 � 5

The time constant of re-reduction of P700+ was determined fromthe kinetics of the absorbance change at 820 nm after far-red illu-mination in intact cells. The kinetic traces were recorded on dark-and light-adapted (5 min, 50 mmol photons m-2 s-1) cells with andwithout incubation with 40 mm CdCl2 for 60 min.Values are given asmeans � SD (n = 3).

Figure 4. Chlorophyll a fluorescence induction transients incontrol (a) (60 min dark adapted) and Cd2+-treated (b) (40 mmCdCl2, 60 min dark incubated) Synechocystis cells. Fo and Fm wereobtained before the actinic light (AL: 50 mmol photons m-2 s-1

red light) was applied. Fm′ values were determined at every thirdminute. After 30 min illumination, 1 mm methyl viologen (MV)was added. Arrows indicate the onset of AL and the addition ofMV.

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maximum at around 30 °C, originates from charge recom-bination between the S2 state of the water splitting enzymeand the reduced secondary quinone acceptor (QB) of PSII.In the presence of DCMU, the so-called Q band can beobserved. This band originates from charge recombinationbetween the reduced primary PSII acceptor QA and the S2

state of the water-splitting enzyme and exhibits a maximumat around 10 °C. Because of the reduced PSII activity inCd2+-treated cells, the amplitudes of both the B and the Qbands decreased parallel during Cd2+ treatment (Fig. 6).Theband positions did not shift suggesting unperturbed redoxpotential of PSII acceptors and donors involved in thecharge recombination process.

In order to check the possible reaction centre (RC)protein degradation, we measured the amounts of PSII RCprotein D1 in control and Cd2+-treated sample at differenttime intervals after the treatment. As shown in Fig. 7, Cd2+

treatment decreased the D1 protein level by about 50%in 5 h.

Effect of cadmium on the photosynthetic activity wasalso recorded under different light conditions. The treat-ment was carried out either in the dark or under growthlight conditions. The whole-chain ET and the PSII-dependent oxygen evolution (H2O → pBQ) were examined(Fig. 8). The fast, short-term inhibition in the NADPH-dependent whole-chain ET was observed equally in thedark and in the light. In contrast, the long-term PSII activitydecrease was avoided in the dark and evolved only in cellsexposed to Cd2+ in the light.

The rate of inactivation of PSII RC under environmentalstresses depends on the balance between the damage and therepair mechanisms of the RC proteins. Chloramphenicolinhibits the protein translation at the ribosomes and allowsfollowing the net protein damage without recovery pro-cesses. We have investigated the effect of chloramphenicolon Fv/Fm, the maximal efficiency of PSII in the presence

and absence of Cd2+ at different light intensities (Fig. 9).Chloramphenicol treatment induced a decrease in Fv/Fm as afunction of time in a light intensity-dependent way.In the presence of Cd2+, the effect of chloramphenicol wasenhanced at all applied light intensities. The loss of PSIIfunction was faster with increasing light intensities and theextent of inhibition was greater than by chloramphenicolalone. Interestingly, Cd2+ decreased the Fv/Fm at the samerate both in the presence and absence of chloramphenicol,suggesting that Cd2+, in addition to damaging PSII, alsoinhibits its repair.

Concentration dependences of different responses to Cd2+

treatment, that is the short-term effect, the fast inhibitionof the whole-chain ET, and the long-term effect, theslow decline of PSII effective quantum yield and theslow decrease in Fv/Fm parameter (PSII maximal quantumyield), were compared. Interestingly, the concentration

Figure 5. Maximal quantum yield of photosystem II (PSII)(Fv/Fm) and the relative amount of the maximal photo-oxidizableP700 (Pm) as a function of time after the addition of 40 mm CdCl2

to Synechocystis cells. Values are given as means � SD (n = 3). Figure 6. Changes of the amplitudes of the B and Qthermoluminescence bands in Synechocystis cells as a function oftime following the 40 mm CdCl2 treatment. The samples wereexcited with a single turnover saturating flash at -20 °C in theabsence and presence of 10 mm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) for the B and Q bands, respectively. Dataare given as percentage of the untreated control. Values are givenas means � SD (n = 3).

Figure 7. Immunoblot analysis of D1 protein in control and40 mm CdCl2-treated Synechocystis cells. The loaded sample ineach lane corresponds to 0.15 mg of total chlorophyll.

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dependences did not differ from each other and the half-maximal inhibition (I50) in all cases required the same Cd2+

concentration, about 20 mm (Fig. 10). The derived I50 values,19.3 � 2.1 (SE, n = 6) for Fv/Fm, 14.8 � 4.1 (SE, n = 5) forFPSII and 20.2 � 3.8 (SE, n = 6) for electron transport rate(ETR), respectively, do not differ significantly at P > 0.05.

DISCUSSION

In photosynthetic organisms, photosynthesis is one of themain targets of the toxic effects of Cd2+ (Nagel & Voigt1995). In plants, the slow uptake rate and delayed

accumulation of Cd2+ may mask the primary effects on thephotosynthetic apparatus. Therefore, the primary and sec-ondary inhibitory effects on photosynthesis are not sepa-rated, resulting in a rather complex inhibitory pattern, withdifferent Cd2+ concentration dependences.

In aquatic plants, algae and cyanobacteria, the cells aremore directly exposed to Cd2+, which is thus capable toinhibit the photosynthetic processes drastically in consider-ably shorter time than in terrestrial plants. Despite the factthat there is no known biological function for cadmium,these photosynthetic organisms take up Cd2+ in consider-able quantity in a relatively short time (less than 5 min) [see

Figure 8. Time courses of the short-term [60 min (a)] and long-term [10 h (b)] effects of CdCl2 on Synechocystis cells incubated in thepresence of 40 mm CdCl2 in the dark and in the growth light (25 mmol photons m-2 s-1 white light). In (a) and (b) respectively, the rates ofthe whole-chain electron transport, H2O→pBQ Hill reaction, were measured. Values are given as means � SD (n = 3).

Figure 9. Long-term effect of cadmium on the Fv/Fm in the presence and absence of chloramphenicol at three different light intensities.Synechocystis cells were incubated in the presence or absence of 40 mm CdCl2 and 200 mg mL-1 chloramphenicol, as indicated at 50 (a),100 (b) and 250 (c) mmol photons m-2 s-1 photon flux density of white light. Values are given as means � SD (n = 3).

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Fig. 2, cf. also on Spirulina platensis (Rangsayatorn et al.2002) and on Synechocystis aquatilis (Pawlik et al. 1993)].Hence, in cyanobacteria the primary effects of Cd2+ toxicitycan be manifested fast enough to discriminate between thefast primary and the slower, secondary effects.

In our experiments, Synechocystis proved to be very sen-sitive to Cd2+ treatment (Fig. 1) and already a few mm Cd2+

drastically decreased the photosynthetic activity after ashort time of incubation (Fig. 2). Comparable sensitivity toour observations was recorded in the marine cyanobacte-rium S. aquatilis (Pawlik et al. 1993) and in the freshwatercyanobacterium Microcystis aeruginosa (Zhou, Juneau &Qiu 2006), where, however, a few mm of Cd2+ was ableto induce significant changes only in 2 h. In some othercyanobacterial species, for example, in Synechococcus,much higher Cd2+ concentrations engendered even weakerresponse (Tumova & Sofrova 2002). Possible reasons forthe lower response are the Cd2+ adsorption on the cell wallor a more effective operation of inactivation mechanisms,for example, by induction of chelator proteins (Cobbett &Goldsbrough 2002). In Scenedesmus armatus and Chlamy-domonas reinhardtii, also lower cadmium sensitivity of pho-tosynthetic processes was detected (Tukaj et al. 2007). Inthese green algae, a few hundred micromolar Cd2+ causedsimilar magnitude inhibition of photosynthesis as in ourexperiments. In the case of higher plants, the applied Cd2+

concentrations often are unrealistically high.By using Synechocystis cells, we could establish the

sequence of different responses to Cd2+. In this organism,the fast uptake and the applied Cd2+ concentrations com-bined with different light conditions ensure the narrow

overlap between different responses provoked by Cd2+. Inthe first step, inhibition of the whole-chain ET can beobserved in a few minutes after the addition of Cd2+ to thecell culture; in about an hour, with 40 mm CdCl2, the CO2-dependent O2 evolution is fully blocked (Fig. 2). Neverthe-less, the cells retain their viability even after 12 h Cd2+

treatment (Supporting Information Fig. S1) despite thecomplete halt of photosynthetic processes and cell division.This shows that suppression of photosynthesis at the Cd2+

concentration applied in our experiments is a specific, directeffect rather than arising from a general decline of cellmetabolism. In the green alga, C. reinhardtii, the predomi-nant proportion of cadmium was localized in chloroplasts(Nagel, Adelmeier & Voigt 1996), supporting the notionthat photosynthetic processes are the prime targets of Cd2+

toxicity. Further, the cadmium tolerant mutant of C. rein-hardtii exhibits an impaired photosynthesis also in theabsence of Cd2+, suggesting that photosynthesis is more sen-sitive target of Cd2+ toxicity than any other metabolicprocess in green algae (Nagel & Voigt 1995).

In S. aquatilis, Cd2+ uptake has been shown to occur viaactive transport (Pawlik et al. 1993). We observed that thefast effect of Cd2+, that is the inhibition of the whole-chainET, occurs at the same rate in the dark as under growth lightintensity (Fig. 8a), suggesting that oxidative phosphoryla-tion can be sufficient for the uptake and it does not demandphotophosphorylation.

The insensitivity of MV-dependent O2 uptake and thePSII-dependent (H2O→pBQ) O2 evolution to Cd2+ (Fig. 2)can rule out the possibility that the primary site of inhibi-tion is localized in PSII or PSI or the intersystem ET com-ponents. These data are consistent with literature data(Sas et al. 2006; Zhou et al. 2006) and show that the fastinhibition of ET must be localized downstream of PSIacceptor side.

The first possible target of Cd2+ in terms of the sequenceof ET is the FNR. Inhibition of FNR by Cd2+ has beenreported by Grzyb et al. (2004), though these results arefrom in vitro measurements and 5 mm Cd2+, resulted in only50% inhibition.Taking into account that in our case, in vivo,a few mm was sufficient for an essentially full inhibition, therole of FNR as the primary target can be ruled out. Thisconclusion is consistent with our results on P700 kineticmeasurements, where the presence of Cd2+ does not causeany change in the initial phase of light-induced P700 oxida-tion assigned to ferredoxin activity (Fig. 3) (Harbinson &Hedley 1993). After far-red illumination, which preferen-tially excites PSI, the re-reduction of P700+ is mainly due toPSI-driven cyclic ET (Chow & Hope 2004). In the dark-adapted state, the rates of cyclic ET determined from theA820 transients were essentially identical in the control andthe Cd2+-treated cells (Table 1). In light-adapted state, therate of re-reduction of P700+ increases because of the accu-mulation of reducing power on the acceptor side of PSI(Cornic et al. 2000). The enhanced acceleration of there-reduction of P700+ in Cd2+-treated cells indicatesenhanced NADPH accumulation, which requires the pres-ence of active FNR.

Figure 10. Cadmium concentration dependences of theinhibition of the maximal (Fv/Fm) and the effective (FPSII)quantum yield of photosystem II (PSII) and of the suppressionof the whole-chain electron transport rate (ETR). The values aregiven as the percentage of maximal inhibition. I50 value refers tothe concentration inducing 50% inhibition. Values are given asmeans � SD (n = 3).

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On the basis of the above observations, it is clear that theprimary site of inhibition must be found beyond PSI, that isat later events of photosynthesis, which include the reduc-tion of CO2 and related processes. Our observation thatCd2+ interferes with the slow phase of the light-inducedoxidation kinetics of P700 (Fig. 3), which is known to berelated to the Calvin–Benson cycle (Harbinson & Hedley1993), is in perfect agreement with this conclusion. It can beassumed that the main target of Cd2+ toxicity is one of theenzymes having a role in CO2 fixation, because some of theenzymes, for example, Rubisco, contain free thiol (–SH)groups, which can interact easily with Cd2+ (Clijsters & Van-assche 1985; Stiborova 1988).The changes in the ratio of themetabolites originating from the Rubisco-catalysed reac-tions observed after cadmium treatment suggest activitydecrease of the enzyme (Siedlecka et al. 1997). These obser-vations are not direct indicators of the Rubisco operation,and the effect of decreased substrate level on the enzymeactivity cannot be excluded. Another possible site ofinhibition can be found in the enzymes of the CO2-concentrating mechanism (CCM), which is also coupled toCO2 fixation. CCM has a basic role in the operation of theRubisco in aquatic photosynthetic organisms as it ensuresthe necessary CO2 in sufficiently high concentrations in themicroenvironment of the enzyme for the domination ofcarboxylation processes (Kaplan & Reinhold 1999). Thekey enzymes of CCM are the CAs, which are localizedtogether with Rubisco in the carboxysomes. They are alsopotential targets to Cd2+, because a Zn ion coordinated in itsactive centre can be replaced by Cd2+ (Aravind & Prasad2004). In vitro studies on human CA showed that the Zn2+

replacement by Cd2+ was accompanied by the loss ofenzyme activity (Bauer, Limkilde & Johansen 1977). In theabsence of the CCM key enzyme, CA, the cells cannotprovide the conditions necessary for the operation of theRubisco (Moroney et al. 1989). Decrease of Rubisco activityat the same rate as the decrease of the CA has been pub-lished in the literature (Pawlik et al. 1993; Siedlecka et al.1997). In our previous work, perturbed angular correlationof g-rays (PAC) spectroscopy measurements have shown invivo that in Synechocystis cells in the presence of 40 mmCdCl2, fast, 20–30 min incubation causes a specific bindingof Cd2+ to a CA like protein (Sas et al. 2006). Further, it hasbeen reported (Lane et al. 2005) that in presence of Cd2+,the synthesis of a novel CA enzyme that contains Cd2+ in itscatalytic centre instead of Zn2+ is induced in the marinediatom Thalassiosira weissflogii; the physiological role ofthis CA is not completely clarified yet but it might indicatethat the Zn2+ containing isoform is sensitive to Cd2+ (Laneet al. 2005).

Until 4 h after the addition of Cd2+, the TL (Fig. 6) andchlorophyll fluorescence transient (Fig. 5) measurementsshow no significant changes in PSII functions, and PSI activ-ity (Fig. 5) remains unaffected even after 12 h of the start ofthe treatment. In contrast to the maximal efficiency, theoperative efficiency of PSII in light-adapted state (FPSII) isdecreasing in the sample pre-incubated with 40 mm CdCl2

(Fig. 4). The reason of this decrease is mainly due to the

reduced number of open reaction centres (increase of theFs), rather than the decrease of the intrinsic maximumquantum yield of PSII (decrease of Fm′). This is supportedby the fact that the PSII effective quantum yield can berestored to the control value by the addition of PSI electronacceptor, MV (1 mm), to the cells. In other terms, theincreased Fs can be explained by the combined effect ofillumination and the block of ET beyond PSI.

In the second phase of the response to Cd2+ treatment,the activity of PSII (the maximal quantum efficiency) andthe primary photochemical processes of PSII (Figs 5 & 6,respectively) show gradual decrease as a function of time.These decreases and the pBQ-dependent O2 evolution(Fig. 8b circle) are manifested after 4 h of incubation in thepresence of cadmium. In the literature, two target sites, atwhich PSII inactivation occurs upon cadmium treatment,have been proposed in higher plants. Several authors sug-gested donor side effect as the origin of the Cd2+-induceddecrease in PSII activity (Krupa et al. 1987; Krupa et al.1993; Faller et al. 2005; Bernat et al. 2011), while others pro-posed the inactivation of the PSII RCs (Atal et al. 1991;Jegerschold et al. 1995). It is also suggested that the photo-activation of PSII RC is inhibited by cadmium at low con-centrations because of a competition between Cd2+ and Ca2+

for the Ca2+-binding site, which hampers the assembly ofPSII with functional donor side (Faller et al. 2005).

Our TL results do not support a specific inactivation ofthe water-splitting complex. Both the B and Q TL bandsdepend on S2 states of the water-oxidizing complex (WOC).Although the parallel decrease of these two bands wouldsuggest the inactivation of donor side, in this case the TyrZand TyrD redox-active residuals of the heterodimer RCproteins would replace the S2 state of water-splittingenzyme in the charge recombination processes, whichshould increase the C band and the after-glow thermolumi-nescence (AT) band originating from TyrZ+/QA

- andTyrD+/QA

- charge recombinations, respectively (Vass 2003);however, neither the appearance of AT band nor theenhancement of C band was observed (Supporting Infor-mation Fig. S3), similar to our previous results (Sas et al.2006). Hence, our TL data reflect the inactivation of RCsrather than specific donor side effect. These results are ingood agreement with the data from chl a fluorescence tran-sient measurements where the same gradual long-termdecrease of Fm was observed both in the absence and pres-ence of DCMU (Supporting Information Fig. S4). Fm inDCMU-treated sample does not depend on the WOC aslong as the RC is active, because a single electron providedby TyrZ or TyrD is sufficient to fully reduce QA. Hence, thedecrease of Fm in the presence DCMU reflects the impair-ment of RCs. Immunoblot analysis (Fig. 7) also shows thatthe amount of D1 protein decreases together with PSIIfluorescence and TL, further supporting our assumptionthat the decrease of PSII activity is due to the inactivationof the RC rather than the impairment of PSII donor side.

In contrast to the short-term effect, which occurs at thesame rate in the dark as in light, the long-term Cd2+-inducedinhibition on PSII activity, that is the decrease of O2

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evolving capacity in the presence of pBQ and the decline ofFv/Fm, was observed in cells that were kept in light after theCd2+ treatment (Fig. 8); the rate and extent of this inactiva-tion of PSII increased with increasing light intensity(Fig. 9). It is well established that even moderate light canpromote ROS formation under stress conditions (Huneret al. 1996). The direct ROS formation ability of Cd2+ viaFenton’s reaction is rather low but it might produce ROSindirectly in the presence of light by blocking the ETbecause of the limitation of CO2 fixation and the increasedexcitation pressure on PSII (Long, Humphries & Falkowski1994; Kornyeyev, Logan & Holaday 2010). This is indicatedby the elevated Fs level that can be alleviated by the addi-tion of MV (Fig. 4) serving as an artificial PSI electronacceptor.

It is generally accepted that the ROS generated understress conditions can directly inactivate the photochemicalreaction centre of PSII (Keren et al. 1997; Vass 2012). Thismodel has, however, been challenged by the scheme inwhich ROS is proposed to inhibit the repair of photodam-aged PSII by suppressing primarily the de novo synthesis ofproteins (Murata et al. 2007; Nishiyama, Allakhverdiev &Murata 2011). The damage of RC can be monitored sepa-rately from its recovery in the presence of protein synthesisinhibitors such as chloramphenicol (Nishiyama et al. 2001).Our observation that Cd2+ enhances the rate of PSII inac-tivation measured both in the presence and absence ofchloramphenicol (Fig. 9) shows that while Cd2+ promotesthe direct inhibition of PSII RC it also inhibits its recovery.As concerns, the limited recovery may be due to theretarded de novo protein synthesis. The last step of PSIIrecovery from photodamage is the photo-activation of theRC. Because it has been shown that in C. reinhardtii Cd2+

directly inhibits this step at low concentrations (Faller et al.2005), we cannot rule out that hampered photo-activationmay also contribute to the lack of efficient recovery inCd2+-treated Synechocystis cells, as well.

Comparison of the concentration dependence of short-and long-term Cd2+ effects followed the pattern andresulted in approximately the same I50 value despite thedifferences in the inhibitory sites and times required for theinhibition (Fig. 10). This strongly suggests that all the aboveeffects originate from the same primary effect. Evidently,the short-term effect, the fast inhibition of ET, must beconsidered the primary inhibitory effect, which then can beheld responsible for the long-term effects, the slowly evolv-ing secondary effects affecting PSII.

Based on the data presented in this work and our pre-vious results, together with data available in the literature,we propose that the multiphase effect of Cd2+ on the pho-tosynthesis of Synechocystis cells primarily stems from asingle site of inhibition and is extended via a cascademechanism according to the following scheme: Cd2+ is ableto penetrate through the cell wall and to accumulate inthe cells even at low environmental concentrations; itinhibits the dark phase of the photosynthesis via specifi-cally binding to (an) enzyme(s) of the Calvin–Bensoncycle and/or that involved in the CCM. Inhibition of

carbon fixation decreases the consumption of ATP andNADPH, which, in turn, in the presence of light, increasesthe ATP/ADP and NADPH/NADP ratios. This engendersthe block of the ET; this, combined with the effect ofsimultaneous illumination, can bring about an elevatedexcitation pressure on PSII. The over-reduction of ETcomponents can increase the probability of charge recom-bination between P680+ and reduced pheophytin thatproduce the excited state of the reaction centre, P680*,and thus can induce the production of ROS (Vass 2011;Vass 2012). ROS accumulation decreases the D1 proteinlevel directly or indirectly by blocking the protein synthe-sis and recovery of the PSII RCs that finally leads to theimpairment of the reaction centre functions. At the sametime, via an independent mechanism, the Cd2+ mayenhance the PSII inactivation by suppressing de novoprotein synthesis and photo-activation of PSII.

ACKNOWLEDGMENTS

This work was supported by grants from the HungarianResearch Fund (OTKA, CNK-8345 and OTKA, 68692), theSocial Renewal Operational Programme (TÁMOP-4.2.2/B-10/1-2010-0012) and Hungary-Romania Cross-BorderCo-operation Programme (HURO/0901/270/2.2.2.)

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Received 21 February 2012; received in revised form 2 May 2012;accepted for publication 6 May 2012

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Effect of Cd2+ on the viability of Synechocystiscells. Cells were incubated without (a) and with 40 mmCdCl2 (b, c), and at various times aliquots were taken andcentrifuged; then the cells were resuspended in Cd-free (aand b) and in 40 mm CdCl2 containing cultivation medium,(c) and equal volumes were loaded into the wells of tissueculture plates filled with agar containing BG11 medium.The photo was taken after 3 d incubation under growthconditions.Figure S2. Re-reduction kinetics of P700+ after far-redillumination in control (•) and Cd2+ pretreated (40 mmCdCl2, 1 h) (•) Synechocystis cells before (a) and after (b)5 min illumination with 37 mmol photon m-2 s-1 actinic light.Arrows indicate the cessation of far-red light.Figure S3. Thermoluminescence glow curves of control(black curves) and CdCl2-treated (red curves) (40 mm CdCl2

for 10 h under growth light) Synechocystis cells. The B band(a) (originating from the S2/QB

- charge recombination) andthe Q band (b) (originating from the S2/QA

- charge recom-bination) were measured in the absence and presence of10 mm DCMU, respectively.The samples were excited with asingle turnover saturating flash at -20 °C.Figure S4. Effect of 40 mm CdCl2 on the chlorophyll a fluo-rescence transient parameters (Fo, Fm and Fv/Fm) as a func-tion of the time of Cd treatment in the absence (a) andpresence (b) of 10 mm DCMU. Values are given asmeans � SD (n = 3).

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