Growth and photosynthetic responses of the bloom

Chemosphere 65 (2006) 1738–1746 www.elsevier.com/locate/chemosphere

Growth and photosynthetic responses of the bloom-forming cyanobacterium Microcystis aeruginosa to elevated levels of cadmium Wenbin Zhou a, Philippe Juneau b, Baosheng Qiu b

a,*

a College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, PR China De´partement des Sciences Biologiques, Universite´ du Que´bec a` Montreál, Montreál, Que´bec, Canada H3C3P8

Received 18 December 2005; received in revised form 19 April 2006; accepted 27 April 2006 Available online 14 June 2006

Abstract Effects of cadmium (Cd) on the growth and photosynthesis of the bloom-forming cyanobacterium Microcystis aeruginosa Ku¨tz 854 were investigated. The growth was markedly inhibited when it was treated with 4 lM Cd. However, the biomass production was almost not influenced after a prolonged exposure at Cd concentrations 62 lM. Chlorophyll content was more sensitive to Cd toxicity than phycobiliproteins at 0.5 lM Cd. However, the decrease of phycobiliproteins was larger than chlorophyll at the highest Cd concentration. A significant increase of Fv/Fm value was observed at Cd concentrations 62 lM. On the other hand, when cells were treated with 4 lM Cd, Fv/Fm was significantly increased after 12 h of treatment but decreased after 48 h. The true photosynthesis was decreased with the increase of Cd concentration at 2 h. However, we noticed a recovery when the treatment was prolonged. After 48 h of exposure at the highest Cd concentration, photosynthetic oxygen evolution was markedly inhibited but dark respiration increased by 67%. Cellular Cd contents were augmented with the increase of Cd concentration. To our knowledge, we have demonstrated for the first time that the inhibitory site of Cd in M. aeruginosa is not located at the PSII or PSI level, but is probably situated on the ferredoxin/NADP+-oxidoreductase enzyme at the terminal of whole electron transport chain. We noticed also an increase of PSI activity, which is probably linked to the enhancement of cyclic electron transport around PSI. We can conclude that the increase of cyclic electron transport and dark respiration activities, and the decrease of phycobiliproteins might be adaptive mechanisms of M. aeruginosa 854 under high Cd conditions. 2006 Elsevier Ltd. All rights reserved. Keywords: Cadmium toxicity; Chlorophyll fluorescence; Microcystis aeruginosa; Photosynthesis; Photosynthetic electron transport; Pigment

1. Introduction The development of human activities and tion has led to an increased accumulation the environment. The principal sources of tion are combustion of fossil fuels, mining

industrializaof metals in metal polluand smelting

Abbreviations: APC, allophycocyanin; BTP, bis–tris propane; Cd, cadmium; CPC, phycocyanin; DCMU, 3-(3 0 ,4 0 -dichlorophenyl)-1,1-dimethylurea; DCPIP, 2,6-dichlorophenol-indophenol; DCPIPH2, reduced 2,6-dichlorophenol-indophenol; DPC, diphenylcarbazide; FNR, ferredoxin/NADP +-oxidoreductase; Fv/Fm, PSII photochemistry efficiency; MV, methyl viologen; p-BQ, p-benzoquinone. * Corresponding author. Tel.: +86 27 6786 1514. E-mail address: [email protected] (B. Qiu). 0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.04.078

activities, release of wastes and sewage waters, and the use of fertilizers and pesticides (Hanikenne, 2003). The release of metals into aquatic ecosystem and toxicity of these metals on phytoplankton species, including cyanobacteria, have received considerable attention over the past 20 years (Robinson et al., 2000). Inhibition by metals of metabolic processes associated with either respiration or photosynthesis ultimately affects the productivity of the aquatic food chain (Prasad et al., 1991). Cadmium (Cd) is one of the major metal pollutants owing to its high toxicity. Furthermore, Cd has a greater solubility in water compared to other metals, which determines its wide distribution in aquatic ecosystems (Lockwood, 1976). Photosynthetic organisms are known to be

W. Zhou et al. / Chemosphere 65 (2006) 1738–1746

highly sensitive to Cd ions (Clijsters and Van Assche, 1985; Das et al., 1997; Pinto et al., 2003). Several studies have been focused on the inhibition of photosynthesis yet the mechanisms of Cd toxic effect on photosynthetic processes have remained for the most part, elusive (Clijsters and Van Assche, 1985; Krupa and Baszynski, 1995). It has been shown that PSI activity was slightly affected by Cd (Baszyn´ski et al., 1980; Clijsters and Van Assche, 1985; Atal et al., 1991; Siedlecka and Krupa, 1996). On the other hand, cadmium is thought to alter PS II activity by acting either on the oxidizing (donor) side (Van DuijvendijkMatteoli and Desmet, 1975; Atal et al., 1991; Sˇersˇenˇ and Kra´l’ova´, 2001), on the reducing (acceptor) side (Singh and Singh, 1987; Atal et al., 1991; Fodor et al., 1996), or without defined location (Siedlecka and Krupa, 1996). Geiken et al. (1998) demonstrated that Cd could alter the activity of oxygen-evolving complex in pea and broad bean and cause ultimately the disassembly of their PSII. The described effects of Cd stress on photosynthesis were mainly investigated in vitro, using isolated chloroplasts or thylakoid membranes, and some of them either could not be observed in vivo or could not be confirmed by later in vitro studies (Clijsters and Van Assche, 1985; HaagKerwer et al., 1999). Cyanobacteria are an ancient, large and diverse group of prokaryotic autotrophs with an oxygenic photosynthesis (Whitton and Potts, 2000). They are widespread and can be found in many different habitats from aquatic to terrestrial ecosystems. The widespread nature of cyanobacteria in different environments makes them useful as indicator of environmental contamination and pollution (Whitton, 1984). Moreover, these prokaryotic organisms may serve as an excellent tool for studying the effect of metals on photosynthesis owing to their close similarity with the chloroplast of higher plants (Lang, 1968). Indeed, it was shown that cadmium is toxic to cyanobacteria, causing severe inhibition of physiological processes such as growth, photosynthesis, and nitrogen fixation (Payne and Price, 1999; Gaur and Rai, 2001; Miao et al., 2005). However, mechanisms of Cd toxicity and resistance are variable depending on the organism and are not yet well understood (Neelam and Rai, 2003). Microcystis aeruginosa Ku¨tz. has been widely recognized as one of the most common bloomforming cyanobacteria found in fresh waters (Reynolds and Walsby, 1975; Oliver and Ganf, 2000), and it has also been found in aquatic ecosystems polluted by heavy metals (Lukacˇ and Aegerter, 1993). The potential for Cd toxicity is often most severe associated with aquatic sediments and interstitial waters because the presence of some metals in high levels has been well documented in sediments (Audry et al., 2004). Microcystis aeruginosa can turn into benthic stages when environmental conditions are harsh and thus can survive for a long time in the sediment. They could recruit to the water column and start to grow when environmental conditions become favorable again (Brunberg and Blomqvist, 2003; Sta˚hl-Delbanco et al., 2003). Lukacˇ and Aegerter (1993) have demonstrated that Cd at

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non-toxic concentrations (0.001–0.20 lM) did not significantly affect the growth and toxin production of M. aeruginosa PCC 7806. Guanzon et al. (1994) have determined the inhibitory effects of copper, zinc and cadmium on the growth and photosynthetic oxygen evolution of three freshwater microalgae, and concluded that M. aeruginosa exhibited higher tolerance than the other two species to metal contamination. Recently, Neelam and Rai (2003) have investigated the interactive effects of UV-B and Cd on the survival, photosynthesis and lipid peroxidation of three cyanobacteria, and found that the damage caused by Cd at 0.27 lM to Microcystis sp. was more pronounced than UV-B (12.9 mW m2 nm1, 35 min). Although many studies have been conducted, the mechanism of Cd toxicity on photosynthesis of M. aeruginosa remains elusive, and to our knowledge no information exists about its adaptation to Cd stress. In order to get new insight on these two aspects, we studied the effect of Cd contamination on pigments concentration, chlorophyll fluorescence, photosynthetic oxygen evolution, dark respiration, electron transport activities and Cd content of M. aeruginosa Ku¨tz. 854 exposed cells. 2. Materials and methods 2.1. Algal material and culture conditions Microcystis aeruginosa Ku¨tz. 854 was provided by the Freshwater Algae Culture Collection of the Institute of Hydrobiology, the Chinese Academy of Sciences. This strain lacks the colony organization when cultured under laboratory conditions. It was grown in BG11 medium (Stanier et al., 1971) at 25 C under continuous illumination of 40 lmol photons m2 s1. Analysis by microscopy established that bacterial biomass in the cultures was negligible. 2.2. Effect of cadmium on cell growth Exponentially grown cells were inoculated in BG11 medium and different concentrations of Cd were applied. The Cd was added as CdCl2 to give final concentrations of 0.5, 1, 2 and 4 lM, corresponding to pCd values of 6.66, 6.36, 6.06 and 5.75. Cultures without Cd addition were used as controls. Chemical speciation of Cd in the exposure solution was estimated using the chemical equilibrium program MINEQL+ (version 3.01b). The formation constants for CdHEDTA1 and CaHCitrate, and the solubility constant for otavite have respectively been modified to 1021.2, 109.33 and 1012.00 according to Twiss et al. (2001). Cell growth was determined by measuring the optical density at 750 nm and the chlorophyll content. 2.3. Measurement of pigments Exponentially grown cells were treated with various Cd concentrations as described above for 48 h. Cultures were sampled respectively at 2 and 48 h for the measurement

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of pigments. Chl a content was determined spectrophotometrically in 80% acetone extracts (Inskeep and Bloom, 1985). The biliproteins were extracted by repeatedly freezing in liquid N2 and thawing at 4 C in the presence of 0.05 M phosphate buffer (mixing equal volumes of 0.1 M KH2PO4 with 0.1 M K2HPO4 solutions, pH 6.7). The homogenized solutions were centrifuged at 4000g for 15 min, and then the absorbance of the supernatant was determined (Siegelman and Kycia, 1978). The concentrations of phycocyanin (CPC) and allophycocyanin (APC) were calculated according to Siegelman and Kycia (1978). The pigments contents were expressed per cell numbers. Cell numbers were determined with a hemocytometer. 2.4. Measurement of chlorophyll fluorescence Exponentially grown cells were treated with various Cd concentrations for 48 h. Samples were taken for chlorophyll fluorescence analysis after 2, 12, 24 and 48 h exposure to CdCl2. Maximum PSII photochemical yield (Fv/Fm) was determined by using a Plant Efficiency Analyser (PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK) with an actinic light of 3000 lmol photons m2 s1. Illumination was provided by an array of six high-intensity lightemitting diodes (with a peak wavelength of 650 nm), which were focused on the sample surface to provide homogeneous illumination over an area 4 mm in diameter. All samples were dark-adapted for 15 min before measurements. 2.5. Measurement of photosynthetic oxygen evolution and dark respiration

Cd concentration. Electron transport activities of intact cells were assayed at 950 lmol photons m2 s1 and 25 C with a Clark-type oxygen electrode as described above. PSII activity was determined by oxygen evolution with H2O as the electron donor and p-benzoquinone (p-BQ) as the electron acceptor in 2 ml BG11 medium containing 25 mM bis–tris propane (BTP, pH 7.8) and 1 mM p-BQ. PSI activity was measured by the light dependent oxygen uptake in the presence of 25 mM BTP (pH 7.8), 0.1 mM 2,6-dichlorophenol indophenol (DCPIP), 5 mM ascorbate (reducing DCPIP to DCPIPH2), 0.1 mM MV, 1 mM NaN3 (inhibiting respiration), 10 lM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (inhibiting PSII activity). The whole electron transport activity was determined by monitoring the light dependent oxygen uptake with H2O as the electron donor and MV as the electron acceptor in 2 ml BG11 medium containing 25 mM BTP (pH 7.8), 1 mM NaN3 (inhibiting respiration) and 0.1 mM MV. The whole electron transport activity in the absence of water-splitting complex was measured by incorporating diphenylcarbazide (DPC) as an electron donor in the assay mixture, which bypasses the oxygen-evolving complex and donates electrons to the PSII reaction center (Yamashita and Butler, 1969). DPC was prepared in acetone and added to the assay mixture at a final concentration of 0.5 mM. The action sites of inhibitor, artificial electron donors and acceptors used in this study are shown in Fig. 1. Their permeability could be judged from the trace of oxygen variation when samples were treated with the above compound alone or together with others in the light or in the dark.

Oxygen evolution was measured with a Clark-type oxygen electrode (Chlorolab 2, Hansatech). Exponentially grown cells were treated with various concentrations of Cd for 48 h. Samples were harvested by centrifugation after 2, 12, 24 and 48 h exposure to CdCl2, and then resuspended in BG11 medium containing the corresponding Cd concentration. Temperature was controlled at 25 C with a Polystat Refrigerated Bath (Cole-Parmer Instrument Co., Vernon Hills, IL, USA). Illumination was provided by a light housing and stabilized power supply (LS2, Hansatech) and light was brought to the surface of the reaction cuvette through a fibre-optic cable (A8, Hansatech). Irradiance was measured with a quantum sensor (QRT1, Hansatech) and neutral density filters were used to obtain 950 lmol photons m2 s1 (400–700 nm). Dark respiration was estimated from O2 uptake by cells incubated in the dark. The rate of true photosynthesis is equal to the sum of oxygen evolution and dark respiration.

2.7. Measurement of cadmium content

2.6. Assay of electron transport activities

Fig. 2 shows the effects of 14 d Cd exposure on the growth of M. aeruginosa. The growth did not appear to be affected at the lowest Cd concentration used (0.5 lM) (P > 0.05, Tukey multiple comparison). However, when Cd concentration was further increased the growth rate tended to be diminished, but this inhibition seems to

Exponentially grown cells were treated with 4 lM Cd for 24 h and cultures without Cd addition were considered as controls. Samples were harvested by centrifugation and resuspended in BG11 medium containing the corresponding

Exponentially grown cells were treated with various Cd concentrations for 48 h. Samples (30 ml) were taken at 2 and 48 h to determine the intracellular metal contents. Cells were harvested by centrifugation (4000g, 15 min) and washed with 15 ml of 2 mM EDTA for 10 min to remove surface-bound metal. After centrifugation, the pellet was digested in 3 ml concentrated nitric acid according to the microwave technique until clear and diluted to 10 ml with deionized water. Cadmium analysis was done by using an inductively coupled plasma atomic emission spectrometry (ICP-AES) (Optima 2000 DV, PerkinElmer instruments, Norwalk, CT, USA). 3. Results 3.1. Growth


hv

hv

H2O OEC

P680

1741

Pheo

O2

QA

QB

DCMU

DPC

PQ p-BQ

Cyt b6/f

PC or Cyt c553

DCPIPH2

P700

Fd

FNR

NADP+

MV

Fig. 1. Schematic representation of photosynthetic electron transport in cyanobacterial cells, showing the action sites of inhibitor and different artificial electron donors and acceptors used in this study. hm, photons of visible light; OEC, oxygen evolving complex; P680, dimeric chlorophyll center which is photooxidized in PSII; Pheo, pheophytin primary electron acceptor of PSII; QA, the quinone secondary electron acceptor of PSII; QB, a plastoquinone bound to PSII which accepts two electron from QA and equilibrates with the thylakoidal plastoquinone-plastoquinol pool; PQ, plastoquinone; Cyt b6/f, cytochrome b6/f complex; PC, plastocyanin; Cyt c553, cytochrome c553; P700, the chlorophyll which is photooxidized in PSI; Fd, ferredoxin; FNR, ferredoxin/NADP+-oxidoreductase.

since chlorophyll concentration decreased but did not have lethal effects (Fig. 2a and b). The chlorophyll content was decreased with the increase of Cd concentration in the medium, and its difference from the control was more evident at late growth phase (Fig. 2b). These suggest that Cd could inhibit chlorophyll synthesis but appears not to affect the biomass production at low Cd concentrations (62 lM) after a prolonged exposure.

Optical density at 750 nm

a Control 0.5 µM 1 µM 2 µM 4 µM

0.1

3.2. Photosynthetic pigments 0.01

Chlorophyll (µg·ml-1)

1

0

2

4

6

8

10

12

14

0

2

4

6 8 Time (d)

10

12

14

b

0.1

0.01

Fig. 2. The growth of Microcystis aeruginosa at various Cd concentrations expressed as optical density at 750 nm (a) and chlorophyll content (b). Data are means ± SD (n = 3).

recover after 14 days of exposure as the growth of M. aeruginosa at Cd concentrations 62 lM showed no difference from the control on the basis of optical density at 750 nm (P > 0.05, Tukey multiple comparison) (Fig. 2a). The growth of M. aeruginosa was severely inhibited and it failed to grow when exposed to 4 lM Cd. After 2 days of Cd exposure, the highest Cd concentration led to the decrease of optical density at 750 nm and chlorophyll content. The results described here showed that Cd reduced the growth rate of M. aeruginosa at high Cd concentrations. Low Cd concentrations (1 and 2 lM) were toxic to M. aeruginosa

Table 1 shows the changes of pigment contents in Cdtreated M. aeruginosa cells. After 2 h Cd exposure, no significant difference was observed for Chl a, CPC, APC, and the ratio of CPC/Chl a and APC/Chl a under various Cd concentrations (P > 0.05, Tukey multiple comparison). The Chl a content per cell was decreased with the increase of Cd concentration in the medium after 48 h treatment. Cadmium could significantly inhibit chlorophyll synthesis even at 0.5 lM Cd (P < 0.05, Tukey multiple comparison). However, the synthesis of phycobiliproteins was not influenced at low Cd concentrations. They remained relatively constant at 0.5 and 1 lM Cd and showed little difference from the control at 48 h (P > 0.05, Tukey multiple comparison). These indicated that chlorophyll synthesis was more sensitive to Cd toxicity than phycobiliproteins under low Cd conditions. High Cd treatments resulted in the decrease of phycobiliproteins content after 48 h exposure. The CPC and APC contents began to decrease in the presence of 2 lM Cd, and decreased significantly at 4 lM Cd (P < 0.05, Tukey multiple comparison). Compared to the control, they were decreased respectively by 68% and 69% at 4 lM Cd. On the other hand, the chlorophyll content in the presence of 4 lM Cd was only decreased by 16%. The ratios of CPC/Chl a and APC/Chl a increased at 0.5 and 1 lM Cd, and then decreased at 2 and 4 lM Cd. 3.3. Chlorophyll fluorescence Fig. 3 shows the changes of maximum PSII photochemical yield when M. aeruginosa was exposed to various Cd concentrations. Interestingly, Fv/Fm value tended to

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Table 1 Pigment contents and pigment ratios of Microcystis aeruginosa exposed to various Cd concentrations for 2 and 48 h Chl a (lg cell1)

CPC (lg cell1)

APC (lg cell1)

CPC/Chl a

APC/Chl a

2h Control 0.5 lM 1 lM 2 lM 4 lM

8.03 ± 0.26 · 108a 8.32 ± 0.30 · 108a 8.02 ± 0.16 · 108a 8.43 ± 0.28 · 108a 8.28 ± 0.26 · 108a

3.34 ± 0.11 · 107a 3.67 ± 0.16 · 107a 3.32 ± 0.15 · 107a 3.68 ± 0.21 · 107a 3.30 ± 0.30 · 107a

6.82 ± 1.77 · 108a 8.22 ± 1.75 · 108a 10.10 ± 4.31 · 108a 7.81 ± 1.74 · 108a 5.70 ± 0.91 · 108a

4.16 ± 0.14a 4.41 ± 0.19a 4.14 ± 0.18a 4.32 ± 0.23a 3.99 ± 0.36a

0.85 ± 0.22a 0.99 ± 0.21a 1.36 ± 0.54a 0.93 ± 0.21a 0.69 ± 0.11a

48 h Control 0.5 lM 1 lM 2 lM 4 lM

9.98 ± 0.53 · 108a 9.15 ± 0.16 · 108b 8.49 ± 0.09 · 108bc 8.79 ± 0.17 · 108bc 8.41 ± 0.14 · 108c

5.09 ± 0.04 · 107a 5.15 ± 0.13 · 107a 4.82 ± 0.06 · 107a 4.14 ± 0.16 · 107b 1.61 ± 0.14 · 107c

9.54 ± 4.26 · 108a 10.07 ± 1.42 · 108a 10.05 ± 0.91 · 108a 8.26 ± 0.86 · 108a 2.96 ± 1.48 · 108b

5.11 ± 0.05a 5.63 ± 0.15b 5.69 ± 0.08bc 4.71 ± 0.18a 1.91 ± 0.16d

0.96 ± 0.43ab 1.17 ± 0.15b 1.24 ± 0.11b 0.94 ± 0.10b 0.35 ± 0.18a

Cd treatments

Data are means ± SD (n = 3). a,b,c Those with different superscript letters in the same column are significantly different (P < 0.05, Tukey multiple comparison).

0.5

Control 2 µM

0.5 µM 4 µM

1 µM

Fv / Fm

0.4 0.3 0.2 0.1 0.0 2h

12 h 24 h Time of Cd exposure

48 h

Fig. 3. Time course of PSII photochemical efficiency (Fv/Fm) of Microcystis aeruginosa up to 48 h exposure to various Cd concentrations. Data are means ± SD (n = 3–4).

increase at 2 h, and a significant increase was observed at 12, 24 and 48 h when M. aeruginosa was exposed to Cd concentrations 62 lM (P < 0.05, Tukey multiple comparison). The variation of Fv/Fm values showed a biphasic pattern when samples were treated with 4 lM Cd. Indeed, Fv/Fm values were significantly increased by 11% and 17% respectively at 2 and 12 h compared to the control (P < 0.05, Tukey multiple comparison). Subsequently, the maximum PSII photochemical yield was close to the control after 24 h and was significantly decreased (21%) compared to the control after 48 h (P < 0.05, Tukey multiple comparison). 3.4. Photosynthetic oxygen evolution and dark respiration Fig. 4 shows the changes of photosynthetic oxygen evolution and dark respiration when M. aeruginosa was exposed to various Cd concentrations. The true photosynthesis of samples treated with 0.5 lM Cd was increased

over the 48-h exposure period. However, the changes of true photosynthesis occurred in a biphasic manner when M. aeruginosa was treated with 1, 2 and 4 lM Cd. It was decreased gradually at 2 h with the increase of Cd concentration from 1 lM to 4 lM and was decreased by 10%, 25% and 50% compared to the control for Cd concentrations of respectively 1, 2 and 4 lM. The true photosynthesis of samples exposed to 1 and 2 lM Cd exhibited a recovery at 12 h, and it was higher than the control at 24 and 48 h (P < 0.05, Tukey multiple comparison). In contrast, the true photosynthesis of sample exposed to 4 lM Cd showed a further decline at 12 h and was decreased by 84% compared to control cells. A recovery was observed at 48 h but the true photosynthesis was only 58% of the control. This suggests that low Cd concentrations did not affect the true photosynthesis but oxygen evolution was inhibited strongly at 4 lM Cd. Dark respiration did not show significant variation after 2 h treatment. It tended to decrease at low Cd concentrations (0.5 and 1 lM Cd) after 2 h exposure. However, dark respiration was increased significantly (67%) compared to the control (P < 0.05, Tukey multiple comparison) when cells were exposed 48 h to 4 lM Cd. 3.5. Photosynthetic electron transport Table 2 shows photosynthetic electron transport activities of M. aeruginosa in the absence or in the presence of 4 lM Cd for 24 h. Interestingly, PSII activity (H2O ! pBQ) was not significantly affected by Cd treatment compared to control cells (P > 0.05, t-test). This suggested that Cd had no inhibitory effect on PSII activity and therefore that PSII was not the action site of Cd. On the other hand, PSI activity (DCPIPH2 ! MV) increased significantly (23%) when Cd was applied (P < 0.05, t-test). The electron transport activities of whole chain were measured in the presence and absence of DPC. When high Cd concentration was present, the electron transport activities from H2O to MV and from DPC to MV were greatly increased by 69%

True photosynthesis Dark respiration -1 -1 (μmol O2·min-1·mg-1 chl) (μmol O2·min ·mg chl)


Control

0.5 µM

1 µM

2 µM

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4 µM

25 20 15 10 5 0 -5 -10 2h

12 h

24 h

48 h

Time of Cd exposure

Fig. 4. Time course of the true photosynthesis and dark respiration of Microcystis aeruginosa up to 48 h exposure to various Cd concentrations. Data are means ± SD (n = 3–4).

Table 2 Photosynthetic electron transport activities of Microcystis aeruginosa Electron transport activity Whole chain (H2O ! MV) Whole chain (DPC ! MV) PSI (DCPIPH2 ! MV) PSII (H2O ! p-BQ)

Rate (lmol O2 h1 mg1 chl)

Table 3 Cadmium contents of Microcystis aeruginosa exposed to various Cd concentrations for 2 and 48 h

Control

Cd treatments

Cd contents (lg cell1) 2h

48 h

Control 0.5 lM 1 lM 2 lM 4 lM

0.23 ± 0.08 · 109a 0.91 ± 0.13 · 109a 2.41 ± 0.80 · 109bc 2.01 ± 0.11 · 109b 3.14 ± 0.13 · 109c

0.09 ± 0.09 · 109a 4.29 ± 0.93 · 109b 5.22 ± 0.82 · 109b 10.49 ± 1.22 · 109c 19.59 ± 2.34 · 109d

339.6 ± 24.3

Cd treatment (100)a

572.8 ± 63.9

(169)b

285.1 ± 53.8

(100)a

475.6 ± 53.1

(167)b

1576.9 ± 128.4

(100)a

1940.8 ± 29.4

(123)b

313.5 ± 19.0

(100)a

341.7 ± 57.2

(109)a

Exponentially grown cells were treated with 4 lM Cd for 24 h and those without Cd addition as the control. Measurements were preformed after samples were harvested and resuspended in BG11 medium containing corresponding Cd concentration. Data are means ± SD (n = 3–4). a,b Those with different superscript letters in the same row are significantly different (P < 0.05, t-test).

and 67% respectively compared to the control (P < 0.05, ttest). The presence of DPC did not modulate the response of whole chain activity to Cd treatment. It suggests that Cd did not affect the oxygen-evolving complex of PSII, and this is in agreement with the result on PSII activity (Table 2). Our results indicate also that Cd treatment had a positive effect on the electron transport chain between PSII and PSI (from PQ to plastocyanin through the cytochrome b6/f complex) because the increase of whole chain activity was much higher than PSI activity and PSII activity showed no obvious changes. However, the photosynthetic oxygen evolution was markedly decreased when M. aeruginosa was exposed to 4 lM Cd for 24 h (Fig. 4). 3.6. Intracellular cadmium content Table 3 shows the Cd uptake in M. aeruginosa exposed to various Cd concentrations for 2 and 48 h. As Cd concen-

Data are means ± SD (n = 3). a,b,c,d Those with different superscript letters in the same column are significantly different (P < 0.05, Tukey multiple comparison).

tration in the external medium increased, there was a concomitant increase in intracellular Cd content. Cellular Cd contents were increased significantly with time at the same Cd concentration (P < 0.05, t-test). There was a sharp rise in intracellular Cd when Cd concentrations in the medium were 2 and 4 lM after 48 h treatment (P < 0.05, Tukey multiple comparison). The Cd contents per cell decreased in the control after 48 h of incubation, presumably due to the dilution effect resulted from cell duplication. 4. Discussion As mentioned in introduction, the effects of Cd stress on the photosynthetic apparatus are still controversial. Neelam and Rai (2003) reported that Cd inhibited PSII and PSI activity when cell-free thylakoid membrane of Microcystis sp. was treated with 0.27 lM Cd. However, our results have clearly shown that PSII and PSI were not the inhibitory sites of Cd in M. aeruginosa and the action site seems to be situated at the terminal part of the electron transport chain (Table 2). To our knowledge, this is the first report showing that Cd stress does not inhibit the PSII

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or PSI activity. De Filippis et al. (1981) have reported that the water splitting system and the FNR (ferredoxin/ NADP+-oxidoreductase) were inhibited in Cd-treated Euglena gracilis. The decrease of FNR activity was also observed in Cd-treated, isolated chloroplasts of 21-dayold maize (Siedlecka and Baszyn´ski, 1993). The FNR is a sulfhydryl requiring enzyme located at the final lightdependent stage of NADPH formation (Forti, 1977). The interaction of metals with the functional SH-groups of proteins is generally proposed as the mechanism for several inhibitory reactions (De Filippis et al., 1981; Clijsters and Van Assche, 1985; Van Assche and Clijsters, 1990). Cd could directly affect the sulfhydryl homeostasis and inhibit SH-bearing redox-regulated enzymes (Hall, 2002; Schu¨tzendu¨bel and Polle, 2002). The major reason for Cd toxicity is probably due to its higher affinity to thiol groups than most metallic micronutrients. In the present study, the photosynthetic oxygen evolution was markedly decreased when M. aeruginosa was exposed to 4 lM Cd for 24 h (Fig. 4). However, we have also shown that the electron transport activity from H2O to MV was greatly increased by 69% compared to the control (P < 0.05, t-test). Therefore, we may advance that Cd affects directly the FNR, but that the decrease in oxygen evolution is due to a feedback mechanism from the Calvin cycle. In the present study, the decrease in chlorophyll and phycocyanin contents observed after 48 h of 0.5 lM Cd treatment (Fig. 2b; Table 1) is consistent with the results of Neelam and Rai (2003), where Cd was shown to inhibit chlorophyll synthesis. Cells treated with high Cd concentrations presented a significant decline in CPC and APC content, and the extent of their decrease was higher than chlorophyll (Table 1). Degradation of the cyanobacterial light harvesting complex, the phycobilisome, is a general acclimation response occurring under various stress conditions (Sendersky et al., 2005). A decline in phycocyanin content due to the adaptation to high levels of zinc had been reported in Synechocystis aquatilis (Andrade et al., 1994). Therefore, the decrease of phycobiliproteins observed in our work might be an adaptive mechanism of M. aeruginosa under high Cd concentrations. The phycobiliproteins are the principal light-harvesting antenna pigments of cyanobacteria, and the light energy absorbed by the phycobilisome is mainly transferred to PSII reaction center (Campbell et al., 1998; Bhaya et al., 2000). A significant decrease was observed in the CPC/ Chl a and APC/Chl a ratios in high Cd-treated M. aeruginosa (Table 1). This could result in the decrease of the rate constant for energy trappings of PSII reaction centers and lead to the distribution of excitation energy in favor of PSI, and hence increases the PSI activity (Jeanjean et al., 1993; Hibino et al., 1996; Lu and Vonshak, 1999). Our data of chlorophyll fluorescence and photosynthetic electron transport clearly support this conclusion. Indeed, Fv/Fm value for samples treated with 4 lM Cd for 48 h was significantly decreased. Meanwhile, high Cd concentrations induced significant increase in the PSI activity

and imposed a positive effect on the electron transport chain between the two photosystems (from plastoquinone to plastocyanin through the cyt b6/f complex) (Table 2). Our results suggest also that the pronounced increase in PSI activity may result from the increase of cyclic electron transport around PSI (where the electrons from PSI are transferred to the plastoquinone pool via Fd, instead of reducing NADP+ to NADPH). During this cyclic process, protons are translocated across the thylakoid membrane and contributed to the establishment of transmembrane proton gradient. This will cause an increase of ATP synthesis but a decrease in the generation of NADPH. The cyclic electron transport around PSI is suggested to play an important role in the generation of ATP, which is required for the adaptation of cyanobacterial cells and plants to stress (Howitt et al., 2001). Several studies have confirmed the increase of cyclic electron flow in cyanobacterial cells under salinity stress (Jeanjean et al., 1993; Hibino et al., 1996; Tanaka et al., 1997; Jeanjean et al., 1998; Howitt et al., 2001) and the stimulation of type-1 NAD(P)H dehydrogenase mediated cyclic electron transport by low CO2 (Deng et al., 2003). Thus, a higher cyclic electron transport activity around PSI could be one of the adaptive mechanisms to Cd stress in M. aeruginosa. On the other hand, a significant increase of dark respiratory activity was observed when M. aeruginosa cells were exposed to the highest Cd concentration for 48 h (Fig. 4). They could provide more energy for the synthesis of stress metabolites and/or stress proteins and then for detoxification in M. aeruginosa during Cd treatment. The bloom-forming cyanobacterium M. aeruginosa was well acclimated to grow under a wide range of Cd concentrations (0.5–2 lM). Cd induced an increase in Fv/Fm over the 48 h exposure to Cd concentrations 62 lM (Fig. 3). The true photosynthesis was decreased with the increase of Cd concentration at 2 h, it was recovered and increased gradually after 2 h of Cd treatments (62 lM Cd) (Fig. 4). These may be related to increased resistance to Cd toxicity. Algae have a variety of mechanisms of metal-stress tolerance, including production of metal binding factors and proteins, synthesis of stress metabolites, exclusion of toxic metals from cells by ion-selective metal transporters, alteration of membrane structure, and excretion or compartmentalization (Gaur and Rai, 2001). The Cd content per cell was increased with the increase of Cd concentration over the 48-h exposure period (Table 3). Certain mechanisms might exist in M. aeruginosa to detoxify the Cdinduced toxicity or repair the Cd-induced damage. The levels of cadmium used in the present study ranges from 0.5 to 4 lM and the free cadmium ion concentration was 43–44% of the total. The highest Cd concentration reported for 2569 U.S. surface waters and 3490 Siberia surface waters was respectively 1.16 and 1.84 lM (Fleischer et al., 1974). Even much higher cadmium concentrations (0.70–4.57 lM) have been found in the Tarapaya River, which is strongly contaminated by mine tailings (Smolders et al., 2003). Experimental studies have shown that phyto-


plankton blooms can remove Cd from solution (Slauenwhite and Wangersky, 1991; Luoma et al., 1998). This process may include adsorption of metals to the new surface area provided by a bloom and/or may involve active metal incorporation into cells. Intracellular uptake would add a new sink for metals during a phytoplankton bloom, and hence enhancing depletion from solution (Slauenwhite and Wangersky, 1991; Luoma et al., 1998). Our results showed that cells of M. aeruginosa are very effective in taking up Cd ion from the external medium (Table 3). This Cd accumulating ability of the bloom-forming cyanobacterium M. aeruginosa can be applied to remove Cd from aquatic environment. The unialgal strain used in this study, M. aeruginosa Ku¨tz. 854, lacks the colony organization in laboratory culturing conditions. However, external mucilage is still present around the cells. The mucilage envelope contains mainly polysaccharides, which might present complexing ligands able to complex Cd (Kaplan et al., 1988). Thus, Cd content might partially be divided inside the cells and also into the mucilage, where this metal has to pass through to reach the cells and possibly be subjected to complexation by the mucilage polysaccharides. In conclusion, the findings reported here suggest that, PSII and PSI were not the inhibitory sites of Cd in M. aeruginosa, and high Cd treatment induced a significant increase in the PSI activity. The increase of PSI activity could result from the enhancement of the cyclic electron transport around PSI. The increase of cyclic electron transport and dark respiration activities, and the decrease of phycobiliproteins content might be adaptive mechanisms developed by M. aeruginosa under high Cd concentrations. Acknowledgements This study was funded by the ‘‘211 Project’’ of Educational Ministry of China, the National Natural Science Foundation of China (No. 30200021), and Wuhan Chenguang Project for Youth Scholar (No. 2004500607124). References Andrade, L., Azevedo, S.M.F.O., Pfeiffer, W.C., 1994. Effects of high zinc concentrations in phytoplankton species from Sepetiba Bay (Brazil). Arq. Biol. Tecnol. 37, 655–666. Atal, N., Saradhi, P.P., Mohanty, P., 1991. Inhibition of the chloroplast photochemical reactions by treatments of wheat seedlings with low concentrations of cadmium: analysis of electron transport activities and changes in fluorescence yield. Plant Cell Physiol. 32, 943–951. Audry, S., Scha¨fer, J., Blanc, G., Jouanneau, J.M., 2004. Fifty-year sedimentary record of heavy metal pollution (Cd, Zn, Cu, Pb) in the Lot River reservoirs (France). Environ. Poll. 132, 413–426. Baszyn´ski, T., Wajda, L., Kro´l, M., Wolin´ska, D., Krupa, Z., Tukendorf, A., 1980. Photosynthetic activities of cadmium-treated tomato plants. Physiol. Plant. 48, 365–370. Bhaya, D., Schwarz, R., Grossman, A.R., 2000. Molecular response to environmental stress. In: Whitton, B.A., Potts, M. (Eds.), Ecology of Cyanobacteria: Their Diversity in Time and Space. Kluwer Academic publishers, Dordrecht, The Netherlands, pp. 397–442.

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