Hg2+, Cu2+, and Pb2+-induced changes in Photosystem II ... - Uqtr

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of the primary acceptors pheophytin or QA should be operative. Hence, we speculate that the energy stor- age data obtained in the presence of metal ions could.
Photosynthesis Research 59: 167–174, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Hg2+, Cu2+ , and Pb2+ -induced changes in Photosystem II photochemical yield and energy storage in isolated thylakoid membranes: A study using simultaneous fluorescence and photoacoustic measurements Nathalie Boucher & Robert Carpentier∗ ´ Groupe de Recherche en Energie et Information Biomol´eculaires, Universit´e du Qu´ebec a` Trois-Rivi`eres, C.P. 500, Trois-Rivi`eres (Qu´ebec), Canada G9A 5H7; ∗ Author for correspondence (fax: + 819-376-5057; e-mail: [email protected]) Received 5 March 1998; accepted in revised form 17 December 1998

Key words: cyclic electron transport, fluorescence, metal ions, Photosystem II, thermal dissipation

Abstract Simultaneous fluorescence and photoacoustic measurements have been used to study the effects of metal ions (copper, lead, and mercury) during dark incubation of thylakoid membranes. The values of the chlorophyll fluorescence parameters Fo (initial fluorescence yield with the reaction centers in the open state), Fm (maximal fluorescence yield), Ft (steady state fluorescence yield) and the calculated parameters, 8o (maximal quantum yield of Photosystem II photochemistry) and 8t (actual quantum yield of Photosystem II photochemistry), strongly decreased in the presence of the metal ions coinciding with an increase in the non-photochemical deexcitation rate constant k(N). It was observed that photosynthetic energy storage measured by photoacoustic spectroscopy also decreased but a large portion of energy storage remained unaffected even at the highest metal ion concentrations used. A maximal inhibition of photosynthetic energy storage of 80% and 50% was obtained with Hg2+ and Cu2+ treated thylakoids, respectively, while energy storage was insensitive to Pb2+ . The results are consistent with the known predominant inhibition of the donor side of Photosystem II by the metal ions. The insensitive portion of energy storage is attributed to the possible recurrence of cyclic electron transport around Photosystem II that would depend on the extent of inhibition produced on the acceptor side by the metal ion used. Abbreviations: Chl – chlorophyll; Fo – initial fluorescence; Fm – maximal fluorescence level; Ft – intermediate fluorescence level; HEPES – (N-[2-hydrxyethyl]piperazine-N0-[2-ethanesulfonic acid]); P680 – primary electron donor of Photosystem II; P700 – primary electron donor of Photosystem I; PAM – pulse amplitude modulated; PAS – photoacoustic spectroscopy; QA – primary quinone acceptor; QB – secondary quinone acceptor; PS I – Photosystem I; PS II – Photosystem II; TyrZ – tyrosine residue of Photosystem II; 8o – maximal quantum yield of Photosystem II photochemistry; 8t – actual quantum yield of Photosystem II photochemistry Introduction Photosynthetic organisms are highly sensitive to heavy metal ions. The effect of metal ions on higher plants includes disruption of many physiological functions by binding to protein sulfhydryl groups and substituting essential ions (Meharg 1994). Several studies have been done to understand their effect on photosynthetic electron transport. A major inconvenience

encountered in the study of metal inhibition is that metals react with the buffer used and with many artificial electron donors or acceptors (Renganathan and Bose 1989, 1990). For these reasons, fluorescence and photoacoustic techniques are appropriate methods to study the effect of metal ions on the electron transport chain because these methods are not invasive and do not require the addition of artificial compounds to measure photosynthetic activity.

168 Variable Chl fluorescence is related to the redox state of the electron acceptor QA and can be used as a probe to study photosynthetic activity (Krause and Weis 1984; Govindjee 1995). The use of pulse amplitude modulated (PAM) fluorometry to monitor various parameters of Chl fluorescence as developed by Schreiber (1987) can give valuable information about the state of the photosynthetic apparatus affected by stress factors (Schreiber et al. 1988). For example, the ratio Fv/Fm, representing the maximum quantum yield of primary photochemistry, is usually reduced by environmental stresses affecting mainly PS II (Krause and Weis 1988). On the other hand, photoacoustic spectroscopy (PAS) is used to monitor the conversion of absorbed energy into thermal dissipation and is also useful to study the effects of environmental stresses affecting electron transport (Charland and Leblanc 1993). In this method, the thermal dissipation from a sample illuminated with an intensity modulated light produces a variation of the gas pressure inside a closed cell. These pressure changes induce sound waves detected by a microphone and then amplified by a lock-in amplifier. Photosynthetic energy storage can be evaluated by comparing heat emission in the presence or absence of saturating non-modulated illumination (Malkin and Cahen 1979; Carpentier et al. 1984). Yet, PAM fluorometry has only been used to study mercury inhibition in cyanobacteria (Murthy et al. 1990) or green alga (Xyländer et al. 1996) and copper action in intact leaves (Lidon et al. 1993; Ouzounidou et al. 1997), and the PAS method to determine copper effects in intact leaves (Ouzounidou et al. 1996; 1997). The purpose of this study is to compare the action of three major metal ions frequently found as pollutants, Cu2+ , Hg2+ , and Pb2+ , in isolated thylakoid membranes. The fluorescence and thermal dissipation parameters were monitored using simultaneous PAM fluorescence and PAS measurements. Major differences in the relative action of these metals on the photochemical yield of PS II and on energy storage are discussed.

Materials and methods Thylakoid membranes isolation. Thylakoid membranes were isolated from spinach leaves as described by Goetze and Carpentier (1990). Thylakoids were resuspended in 50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol and 2 mM MgCl2 . Chlorophyll concentration was determined according to Porra et al. (1989).

Fluorescence and photoacoustic measurements. Thylakoid membranes at a concentration of 200 µg Chl per ml were incubated 15 min at 4 ◦ C in the dark in the presence of selected concentrations of heavy metals and 1 ml was aspirated onto a nitrocellulose filter (Millipore, AA type, 0.8 mm pore size). The filter was cut to the proper dimensions and used in the PA cell (MTEC Photoacoustic cell connected to an EG&G Princeton Applied Research Model 5210 lock-in amplifier) to obtain simultaneous fluorescence and photoacoustic measurements as described previously (Allakhverdiev et al. 1994; Yahyaoui et al. 1997). For photoacoustic measurements, the wavelength, provided by a 150-W Xenon lamp (ILC Technology, Sunnydale, CA), was set at 680 nm by a monochromator (Photon Technology International Inc. Model PT101-001SF) and the beam was modulated at 35 Hz using a mechanical chopper (Scitec Instrument). The latter beam was introduced into an arm of a quadrifurcated fiber-optic guide. The light intensity was controlled with neutral density filters. Two other arms of this fiber optic guide were connected to a PAM-101 (Walz, Effeltrich) for Chl fluorescence measurements. The excitation beam was set at 650 nm by a light-emitting diode (LED, type USBR). The detector, consisting in a PIN-photo-diode (type S 1723) and of a long-pass filter (RG 9) permitted detection of light emission over 700 nm. The last arm of the fiber optic guide was used to provide a non-modulated saturating background illumination (180 W/m2 ) from a Walz KL1500 illuminator. Fluorescence and thermal dissipation parameters. The weak red excitation beam of the PAM-101 provided the Fo level of Chl fluorescence consisting in initial fluorescence with all PS II reaction centers in the open state (Krause and Weis 1984, 1991). At t = 1s, an intermediate level of Chl fluorescence Ft was obtained with the monochromatic modulated light of 680 nm serving as actinic illumination for the fluorescence measurements and used to induce the corresponding photoacoustic signal Qt. The saturating light of the KL1500 illuminator provided the maximal levels of fluorescence and thermal dissipation Fm and Qm, respectively. The ratio Fv/Fm, in which Fv represents the maximal variable fluorescence calculated as (Fm – Fo), reflects the maximal photochemical efficiency, 8o , of photosystem II (Havaux et al. 1991). The actual photochemical efficiency of Photosystem II, 8t , calculated as (Fm – Ft)/Fm, provided a parameter that can be directly correlated with the energy

169 storage parameter measured, (Qm – Qt)/Qm × 100% (Allakhverdiev et al. 1994; Markovic and Carpentier 1995).

Results The effects of Hg2+ , Cu2+ ,and Pb2+ on isolated thylakoid membranes were studied using simultaneous measurements of variable thermal and fluorescence dissipation of absorbed energy. This approach allowed direct comparison of energy storage and variable Chl fluorescence emission because all the parameters studied at a given metal concentration were obtained simultaneously from the same sample. The effect of the metal ions on the fluorescence parameters Fo, Ft, and Fm are shown in Figure 1 together with the effect on the calculated values of 8o (= (Fm – Fo)/Fm) and 8t (= (Fm – Ft)/Fm) that represent the maximal quantum yield of PS II photochemistry (with the reaction centers in the open state) and the actual yield of PS II photochemistry in the presence of the modulated (photoacoustic measuring) actinic beam (with part of the reaction centers in the closed state), respectively (Havaux et al. 1991; Krause and Weis 1991). All the above parameters declined with increasing metal ions concentrations. A decline of variable Chl fluorescence in the presence of Hg2+ , Pb2+ , or Cu2+ was shown on several occasions for thylakoid membranes or Photosystem II -enriched submembrane fractions (Hsu and Lee 1988; Renganathan and Bose 1989; Rashid and Popovic 1990; Bernier et al. 1993; Renger et al. 1993; Arellano et al. 1995). However, 8o is obtained from the parameters Fm and Fo and a study of the correlation between the loss of variable fluorescence and the possible variation of Fm and Fo at various metal ion concentrations has not been presented. It is readily observable from Figure 1 that the decline of 8o is closely correlated with a strong decline of Fm produced in the presence of metal ions but not with that of Fo. Indeed, Fo was not very much affected by the metal ions. A small increase of Fo was seen in the presence of the lowest Hg2+ and Pb2+ concentrations used. A similar behavior of Fo in the presence of Hg2+ was reported for cyanobacteria (Murthy et al. 1990). At the highest metal ion concentrations used, Fm decreased by 75% for Hg2+ , Cu2+ , and Pb2+ while Fo declined only by 25%, 40%, and 20%, respectively. The decline of Fm was more pronounced than the one observed for Fo and generally occurs at lower metal concentrations.

Part of 8o remained even at the highest metal concentrations used where it was clear that the metal ions had reached their maximal effect (see Figure 1). This portion was about 30–35% of the initial value of 8o in the case of Cu2+ and Pb2+ and 10–15% for Hg2+ . The photochemical yield of PS II in the presence of the modulated actinic beam (35 Hz, 680 nm, 10 W/m2 ) also decreased with increasing concentrations of Hg2+ , Cu2+ , and Pb2+ (Figure 1). The decline of 8t was similar to the decline of 8o in terms of metal concentration dependency when Cu2+ or Pb2+ was used. However, in the case of Hg2+ , 8t was affected at much lower concentrations. It is clear from Figure 1A that this is due to the relatively high fluorescence levels Ft, close to Fm, obtained under actinic illumination in the presence of low Hg2+ concentrations. In Figure 1, 8o and 8t decreased with increasing metal ion concentrations due to inhibition of PS II photochemistry. The salt concentration needed to provide an inhibitory effect is lower for Hg2+ than for Pb2+ or Cu2+ indicating that Hg2+ is the most toxic metal inhibiting PS II photochemistry in the isolated thylakoids. However, by comparison with the previous literature, two to ten times higher metal concentrations were required to inhibit electron transport in this study (Samson et al. 1988; Samson and Popovic 1989; Rashid and Popovic 1990; Yruela et al. 1993; Schröder et al. 1994). The requirement for such concentrations in the present study is a result of the filtration procedure required during the sample preparation in order to obtain a condensed film onto the nitrocellulose filters used to measure simultaneous photoacoustic and fluorescence signals. It was already observed by Carpentier et al. (1989) that higher concentrations of inhibitors or artificial electron acceptors were needed to affect PS II membranes in this type of samples. The filtration procedure removes most of the buffer solution and dramatically increases the actual Chl/inhibitor ratio during measurements; hence, higher inhibitor concentrations are required in the remaining buffer solution. The data in Figure 1 can be used to approximate the deexcitation rate constants k(P) and k(N) for the photochemical and non-photochemical deexcitation, respectively, 1/Fo – 1/Fm being proportional to k(P) and 1/Fm proportional to k(N) (Havaux et al. 1991). As reported in Figure 2, photochemical deexcitations are greatly reduced in the presence of metal ions whereas k(N) is dramatically increased. This latter increase was relatively less important for Pb2+ in comparison with the two other metal ions.

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Figure 1. Effect of metal salts on the Chl fluorescence parameters Fo, Ft, and Fm (A, B, C) and on 8o and 8t (D, E, F) in isolated spinach thylakoid membranes. The samples (200 µg Chl/ml in 50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol, and 2 mM MgCl2 ) were dark incubated 15 min in the presence or absence of metal salts, then aspirated onto a nitrocellulose filter. The results represent the mean of three independent experiments. Other details are presented in Materials and methods.

Figure 2. Effect of metal salts on the deexcitation rate constants k(P) and k(N) approximated from Chl fluorescence parameters obtained from Figure 1 as described in the text. Conditions are described in Figure 1.

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Figure 3. Effect of metal salts on energy storage in isolated spinach thylakoid membranes measured by photoacoustic spectroscopy simultaneously in the same samples as in Figure 1. Conditions are described in Figure 1.

Photosynthetic energy storage measured by photoacoustic spectroscopy in the presence of various metal ion concentrations is illustrated in Figure 3. This parameter represents the proportion of absorbed energy that is not released as heat in photochemically active samples but stored into chemical intermediates. The ratio (Qm – Qt)/Qm declined with increasing Hg2+ or Cu2+ concentrations but it became obvious that a large portion of the energy storage activity could not be inhibited by the metals. About 25% energy storage remained with Hg2+ and 50% with Cu2+ . Strikingly, in the presence of Pb2+ , energy storage remained practically unaffected at the concentrations where 8o and 8t were decreased by at least 70% showing that an energy storing photochemical activity remained. The relation between 8t and energy storage is better illustrated in Figure 4. These two parameters can be directly compared because they are calculated from the same photochemical states of the same sample (8t = (Fm – Ft)/Fm and energy storage = (Qm – Qt)/Qm). The data in Figure 4 can be fitted with linear trendlines. For Hg2+ and Cu2+ , energy storage was proportional to 8t even though it became apparent that when 8t = 0, about 20% and 30% of the energy storage remains, respectively. As described above, the energy storage seems not affected by Pb2+

Discussion In this study, the inhibitory action of three metal cations, Hg2+ , Cu2+ and Pb2+ was compared. Because all measurements were performed simultan-

Figure 4. Correlation between energy storage and 8t at various metal salt concentrations. The data were obtained from Figures 1 and 3.

eously in the same sample, the parameters of fluorescence and thermal dissipation obtained at various metal concentrations can be directly compared. From the concentration dependencies of Fm and Fo (Figure 1), it can be readily observed that the decline of 8o is mostly due to a decrease of Fm. Such decrease is usually associated with non-photochemical quenching of Chl fluorescence. However, the present measurements are performed in dark-adapted material where non-photochemical quenching could not have developed. Inhibition on the donor side of PS II was

172 frequently shown to lead to a strongly decreased Fm during fluorescence induction experiments due to the lack of electrons available to provide for the accumulation of photoreduced QA (Govindjee 1995). Similar conclusions are reached here. The reduced Fm (and Ft) values are likely due to the inhibition of the donor side of PS II by the metal ions. In fact, all three metals were previously shown to affect the oxygen evolving complex causing the release of extrinsic polypeptides associated with the stabilization of the Mn cluster implicated in water splitting together with Ca2+ and Cl− ions required as cofactors (Rashid and Popovic 1990; Bernier et al. 1993; Rashid et al. 1994; Bernier and Carpentier 1995; Sabat 1996). Furthermore, Cu2+ was shown to affect electron transport between TyrZ and P680+ , this inhibitory site being predominant at elevated Cu2+ concentrations (Renger et al. 1993; Schröder et al. 1994; Jegerschöld et al. 1995 Hg2+ and Cu2+ also inhibit the acceptor side of PS II. In the case of Cu2+ , it was proposed that this metal ion affects the herbicide and QB binding sites (Mohanty at al. 1989; Renger et al. 1993), alters the binding properties of the non-heme iron located between QA and QB (Jegerschöld et al. 1995), or stabilizes the charge separated state P680+ -Pheo− -QA − due to neutralization of the negative charge on QA − following binding of Cu2+ at the acceptor side of PS II (Yruela et al. 1996). On the other hand, no inhibitory effect of Pb2+ was reported in PS II other than its action on the oxygen-evolving complex. This coincides with a nearly complete reversal of inhibition, even at high concentrations of this metal, with the artificial PS II electron donor diphenylcarbazide (Rashid and Popovic 1990). It should be noted that another site of metal binding is supposed to be located in the light-harvesting complexes. This is evidenced by the decreased Fo values (Figure 1). The decrease of Fo proceeds at higher metal ion concentrations in comparison with the decrease of 8o and 8t (see Figure 1) and is thus related to a different process. A decreased Fo in the presence of Hg2+ was interpreted by structural changes in the antenna pigments (Murthy et al. 1990). Alternatively, it was proposed that non-specific quenching of antenna Chl excited states in the presence of Cu2+ could be due to an increased intersystem crossing rate from the singlet to the triplet state owing to the paramagnetic properties of the heavy metal ions (Arellano et al. 1995; Yruela et al. 1996). In earlier studies, no change in Fo values was observed in Dunaliella tertiolecta or in chloroplasts exposed to Cu2+ (Samson et al. 1988;

Hsu and Lee 1988). However, in those studies Fo was not precisely estimated. Measurements of variable Chl fluorescence have shown that part of 8o and 8t remains even at the highest metal ion concentrations used (Figures 1 and 2). This residual portion was only 10–15% of the control value for Hg2+ but as high as 30–35% for Cu2+ and Pb2+ . Renganathan and Bose (1989) and Arellano et al. (1995) reported that a residual Fv was not suppressed by Cu concentrations that produced a full inhibition of oxygen evolution; Samson and Popovic (1989) reached a similar conclusion for Hg2+ . These authors also suggested that Cu2+ and Hg2+ inhibited the primary photochemistry of only part of the PS II centers while another PS II population was insensitive to these metals. In the present study, however, it is also shown that photosynthetic energy storage measured by photoacoustic spectroscopy is even less reduced than the photochemical yield of PS II (Figure 4). The proportion of unaffected energy storage increased in the order Hg2+ < Cu2+ < Pb2+ . Ouzounidou et al. (1993) reported a similar effect with Cu2+ showing that oxygen evolution in intact leaves was more sensitive than photosynthetic energy storage to Cu2+ . This result was interpreted by an increased cyclic electron transport by the less inhibited PS I (Ouzounidou et al. 1993). It is indeed largely accepted that PS I electron transport is less sensitive to metal ion inhibition in comparison to PS II as it is also the case for several other stresses such as photoinhibition or heat stress (Baszynski et al. 1988; Boucher and Carpentier 1993; Carpentier 1997). Cyclic electron transport around PS I was previously suggested to explain the remaining energy storage activity in leaves affected by heat stress (Havaux et al. 1987; Havaux 1993; Bukhov et al. 1996) or SO2 (Veeranjaneyulu et al. 1991). In support of this hypothesis, PS I photochemistry in intact leaves, measured as photochemical energy storage in far-red light and P700 photooxidation detected as the far-red light induced absorbance change at 820 nm, was more tolerant to Cu2+ compared to the PS II activity (Ouzounidou 1996; Ouzounidou et al. 1997). Less energy storage activity in PS I should remain in the presence of Hg2+ , the latter being an inhibitor of electron transport at the level of plastocyanine (Trebst 1980). However, Carpentier et al. (1990) have suggested that the energy storage measured in isolated thylakoid membranes originates from the reduction of the plastoquinone pool during linear or cyclic electron transport. In a recent report, it was shown that energy

173 storage in PS I-enriched submembrane fractions was only detected when an appropriate exogenous electron donor system was used to keep the electron transport intermediates in a reduced state (Velitchkova and Carpentier 1994). Unlike in intact leaves where endogenous electron donors may be present, cyclic electron transport around PS I in isolated thylakoids likely remains inoperative in the absence of PS II activity because reduced intermediates on the acceptor side of PS I should be quickly oxidized by dissolved oxygen. Energy storage was also less affected by photoinhibition and thermal stress in comparison with photochemical quenching of Chl fluorescence or variable fluorescence in isolated photosynthetic membranes (Lapointe et al. 1993; Yahyaoui et al, 1997). The remaining energy storage was suggested to originate from cyclic electron transport activity in PS II rather than from PS I. Cyclic Photosystem II may involve the photoreduction of cytochrome b559 by acceptor side components of PS II such as QA (Allakhverdiev et al. 1997 and refs. therein) and could be more significant in PS IIβ centers (Lapointe et al. 1993). Thus, the major part of energy storage that remains insensitive to metal ions in thylakoid membranes could originate from cyclic electron transport in PS II. To provide an active PS II cyclic electron transport, the acceptor side of the photosystem should remain unaffected by the metals or at least the photoreduction of the primary acceptors pheophytin or QA should be operative. Hence, we speculate that the energy storage data obtained in the presence of metal ions could indicate the level of acceptor side integrity following inhibition by each specific metal. The above would coincide with the current interpretation of the inhibitory action of Cu2+ on the acceptor side of PS II supporting that charge separation between P680+ and QA − is not fully inhibited by this metal (Schröder et al. 1994; Yruela et al. 1996) thus allowing the reduction of electron transport intermediates involved in cyclic electron transport. Jegerschöld et al. (1995) have observed the formation of a new radical that may reduce P680+ during illumination in the presence of Cu2+ . Finally, the insensitivity of energy storage to Pb2+ could indicate that this metal ion does not affect the acceptor side of PS II as discussed above. Further studies will be required to investigate more precisely the nature of the energy storage reaction(s) remaining in the presence of metal ions.

Acknowledgements This work was supported by Natural Science and Engineering Research Council of Canada (NSERC). N.B. was the recipient of a post-graduate scholarship from NSERC. The authors thank A. Tessier and J. Harnois for their professional research assistance and Dr L. Lorrain for help in some of the experiments.

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