Äîêóìåíò âçÿò èç êýøà ïîèñêîâîé ìàøèíû. Àäðåñ îðèãèíàëüíîãî äîêóìåíòà : http://library.biophys.msu.ru/matorin/3394.pdf Äàòà èçìåíåíèÿ: Mon Dec 14 11:24:28 2009 Äàòà èíäåêñèðîâàíèÿ: Mon Oct 1 19:52:21 2012 Êîäèðîâêà:

ISSN 0006 3509, Biophysics, 2009, Vol. 54, No. 4, pp. 481­485. © Pleiades Publishing, Inc., 2009. Original Russian Text © T.K. Antal, E.E. Graevskaya, D.N. Matorin, A.A. Volgusheva, V.A. Osipov, T.E. Krendeleva, A.B. Rubin, 2009, published in Biofizika, 2009, Vol. 54, No. 4, pp. 681­687.

CELL BIOPHYSICS

Assessment of the Effects of Methylmercury and Copper Ions on Primary Processes of Photosynthesis in Green Microalga Chlamydomonas moewusii by Analysis of the Kinetic Curves of Variable Chlorophyll Fluorescence
T. K. Antal, E. E. Graevskaya, D. N. Matorin, A. A. Volgusheva, V. A. Osipov, T. E. Krendeleva, and A. B. Rubin
Biological Faculty, Moscow State University, Moscow, 119991 Russia e mail: matorin@biophys.msu.ru
Received September 3, 2008; in final form, March 25, 2009

Abstract--The effect of methylmercury and copper ions on the kinetics of light induction and dark relaxation of the variable fluorescence of chlorophyll a has been studied on cultures of unicellular alga Chlamydomonas moewusii. Methylmercury was effective at much lower levels. The toxicants at concentrations that did not decrease the photochemical activity of PS II (Fv/Fm) did affect the electron transport on the acceptor side of PS II, nonphotochemical quenching of excitation in the antenna, and reoxidation of the quinone pool. Our results indicate that this approach can be used for detecting the changes in plant cells at the early stages of toxicant action. Key words: Chlamydomonas moewusii, copper sulfate, methylmercury, fluorescence induction curve, photo system, biotesting DOI: 10.1134/S0006350909040149

INTRODUCTION Technological pollution of water basins with heavy metals affects the phytoplankton, which is the primary producer determining the state of the whole aquatic ecosystem. For algae, the most toxic pollutants are mercury and copper compounds [1]. Heavy metal ions exert their effects by binding with organic acids or phosphate anions, blocking essential (e.g. sulfhydryl) groups, and substituting for other metals in proteins [2­4]. The sequelae of these events are lipid peroxidation, impairment of ion transport and homeostasis, upset ATP balance, enzyme inhibi tion, and DNA damage [5­7]. Mercury compounds are known to suppress the light [1, 8­12] and dark steps [13] of photosynthesis, mainly by hindering membrane processes through interaction with SH compounds and protein disulfide groups as well as by displacing enzyme cofactors [9]. Copper ions at higher concentrations inhibit electron transport in photosystem (PS) II at both donor and acceptor sides [14­15]. In studies of the primary reactions of photosynthe sis and of the photosynthetic apparatus as a whole, widely used are fluorescence methods, which are highly sensitive and allow rapid assessment of a num ber of characteristics such as the concentration of photosynthetic pigments, the photochemical energy

transduction capacity of PS II, the probability of elec tron transport from PS II to the plastoquinone (PQ) pool, and the pH dependent energy dissipation in the PS II antenna [16­19]. Estimation of the maximal quantum yield of photochemical energy transduction from the relative value of variable fluorescence Fv/Fm is a widespread means of assessing the functional state of PS II in various conditions, including the presence of toxicants [20­21]. Thus a decline in Fv/Fm is indica tive of PS II degradation, i.e. appearance of centers incapable of QA reduction. Correct measurement of this parameter requires prior dark adaptation of the specimen, providing maximal QA oxidation and relax ation of the nonphotochemical quenching processes. However, in some cases, e.g. at early steps of toxic action or at low toxicant concentrations, Fv/Fm may change only slightly if at all. In this case it may be use ful to analyze the kinetics of light induction and dark decay of variable fluorescence, which affords informa tion on the changes in electron transport at both PS II sides and through the PQ pool as well as on the pH dependent energy dissipation [22­25]. The reactions of photosynthetic electron transport can be quite sen sitive to external influences, and changes therein sometimes precede PS II destruction. Thus a marked decrease in the rate of electron transport from PS II to the PQ pool is not necessarily accompanied by a

481


482 0.8 0.6 0.4 0.2 Fv/F
m

ANTAL et al. (a)

0 0.8 0.6 0.4 0 (b)

20

40

60

80

20

40

60

80 Time, h

Fig. 1. Dynamics of the relative value of chlorophyll vari able fluorescence (Fv/Fm) during incubation of C. moe wusii under light (30 µE m­2 s­1) in the control (filled circles) and in the presence of (a) MeHg (empty circles, 10­7 M; dark triangles, 5 â 10­7 M; empty triangles, 10­6 M) or (b) Cu2+ (empty circles, 10­6 M; dark trian gles, 10­5 M; empty triangles, 5 · 10­5 M). Prior to fluo rescence assays, the specimens were kept in the dark for 5min.

Chlorophyll fluorescence assays. The Fv/Fm parame ter in the algal suspension was determined using the pulse fluorimeter built at the Chair of Biophysics of the Biological Faculty, MSU for measurements on natural low cell density samples. The standard devia tion calculated from data on four independent speci mens did not exceed 8%. The dark relaxation kinetics was recorded in a stan dard procedure using a PAM 2000 fluorimeter (Walz, Effelrich, Germany). Fluorescence was excited with modulated low intensity light (0.1 E m­2 s­1, 655 nm, 600 Hz). To convert the PS II centers into the closed state with fully reduced QA, the specimen was illu minated for 2 s with intense white light (540 E m­2 s­1) from a halogen lamp (KL 1500, Schott, Germany). Four kinetic curves taken from four separate speci mens were averaged, and the resulting curve was sub jected to three exponential fitting with SigmaPlot 9.0 (Systat Software). The light induction kinetics was registered with Plant Efficiency Analyser (PEA) (Hansatech, King's Lynn, Norfolk, UK), which excites fluorescence with a powerful pulse at 650 nm; in our experiments, the pulse lasted 6 s at a flux density of 3000 E m­2 s­1. RESULTS AND DISCUSSION The Fv/Fm value characterizes the content of pho tochemically active PS II centers, which in its turn reflects the current balance between their light destruction and repair [14, 15]. These measurements were made during 72 h incubation of C. moewusii with different concentrations of MeHg or copper ions. As shown in Fig. 1, MeHg at 10­7 M slightly decreased the Fv/Fm (from 0.7 to 0.6 in 24 h) with further full recovery, indicating cell adaptation to the toxicant. At 5 · 10­7 M, MeHg completely inactivated PS II, which is in line with its reported high toxicity for algae [1, 10]. Copper ions affected our object only at much higher concentrations; 5 · 10­5 M Cu2+ decreased the Fv/Fm from 0.7 to 0.54 in 24 h, and the value did not further change through the observation period, sug gesting equilibration of PS II destruction and repair. It is known that the kinetics of induction of variable fluorescence measured in the millisecond range under saturating light comprises three phases of growth (OJIP) and subsequent decay (see Fig. 2a) [26]. The rise from the minimal (O or Fo) to the maximal level (P or Fm) mainly reflects the transition of PS II centers from the open state with oxidized QA to the closed state with reduced QA. The OP amplitude, just as Fv/Fm, corresponds to the content of photochemically active PS II center capable of QA reduction. The OJ phase reflects partial QA reduction while the PQ pool remains largely oxidized. The following JIP segment reflects the reduction of the remaining QA with com plete reduction of the PQ pool. The relative amplitude
BIOPHYSICS Vol. 54 No. 4 2009

decline in Fv/Fm, e.g. upon the action of herbicides like diuron or atrazine. It should be noted that such kinetic measurements can be run with well known commercially available instruments and take just a few seconds. To see whether such analysis can be expedient in detecting the low dose effects of heavy metals, here we have examined the characteristics of primary photo synthetic processes in microalgal (Chlamydomonas moewusii) cells exposed to methylmercury chloride and copper sulfate at concentrations that only slightly affect Fv/Fm. EXPERIMENTAL Algal culture and toxic exposure. An algologically pure culture of the green unicellular alga C. moewusii Gerloff (Lewin 1002, CALU 228 flagella less mutant), obtained from the collection of the Chair of Microbiology, St. Petersburg State University, was grown phototrophically at 25°C in a Tris acetate phosphate medium (pH 7.0) under artificial daylight (30 E m­2 s­1, 14/10 h light/dark). Aliquots of sta tionary culture (25 mln cells in 100 mL) were trans ferred aseptically into 250 mL flasks, CuSO4 · 5H2O or MeHg chloride (Aldrich) were added at different concentrations, and the culture was further incubated under standard conditions up to 3 days.


ASSESSMENT OF THE EFFECTS OF METHYLMERCURY AND COPPER IONS 2.5 (a) 2.0 1.5 1.0 0.5 (F ­ Fo)/F
o

483

2 I

P MeHg

(b) I 1

2 4

P 3

2.5 2.0 1.5

4

J 1 3 J

0 2.5 2.0 1.5 1.0 0.5 0

O (c) 1 2 CuSO4 3 (d) 2

0.5 2.5 1 2.0

3 4 0.1 1.0 10 100 1000 10 Time, h

4

1.5

100

1000

1.0

Fig. 2. Light induced chlorophyll fluorescence transients (OJIP) measured in C. moewusii before (1) and after (2) 4, (3) 24, and (4) 72 h of incubation under light with (a, b) 10­7 M MeHg or (c, d) 5 · 10­5 M Cu2+. Left panels (a, c) show variable fluorescence (F ­ Fo) relative to Fo; right panels (b, d), relative to (FJ ­ Fo) where FJ is the fluorescence at 3 ms of illumination (inflection J on the curve). Prior to fluorescence assays, the specimens were kept in the dark for 5 min. Presented are typical curves chosen from measurements on four separate specimens using the PEA instrument.

of JIP characterizes the probability of electron trans port from QA to the PQ pool [18], and its decrease tes tifies to impairment of electron transport. The time of reaching the maximal fluorescence yield P(Fm) depends on the PQ pool reduction rate, and its increase testifies to impairment of electron transport [22]. The decline in fluorescence observed for several seconds after reaching the P level reflects nonphoto chemical quenching caused by pH dependent energy dissipation into heat in the PS II antenna [27]. The amplitude of this phase can indirectly characterize the pH magnitude. Figure 2 shows the OJIP transients recorded in C. moewusii cells before and after 4, 24, and 72 h of incu bation with (a) 10­7 M MeHg or (c) 5 · 10­5 M Cu2+ (the concentrations slightly affecting Fv/Fm, see above). One can see that the toxicants attenuated the OP amplitude (Fv/Fo) but the shape of the curves changed little. To estimate the JIP contribution, the curves were also normalized to the OJ amplitude (Fig. 2b,d). MeHg caused a small increase of JIP in 4 h, which could be due to hindered reoxidation of the PQ pool. Indeed, we have recently shown that dibromothymo quinone, an inhibitor of plastoquinone oxidation, slightly increases the maximal fluorescence yield (P) [28]. These results are in accord with the earlier data on the interruption of electron transport between PQ and PS I in the presence of MeHg [12]. The decrease
BIOPHYSICS Vol. 54 No. 4 2009

in the OP amplitude observed in 24 h with MeHg indi cates degradation of PS II centers. By 72 h the OP was restored to the initial level (a), but the JIP contribu tion decreased by 24% (b) and the time to P(Fm) increased from 120 to 180 ms, indicating recovery of PS II photochemical activity and simultaneous appearance of centers with electron transport impaired in the acceptor part. In Fig. 2b one can also see that in 72 h with MeHg there was also an increase in the qE component of nonphotochemical quench ing, which could reflect a greater transmembrane pH. The latter can be caused by disturbed photo phosphorylation in chloroplasts, which is also in line with published data [12]. Thus, prolonged incubation with 10­7 M MeHg did not cause PS II degradation but hindered the electron transport in PS II, decreased PQ reoxidation, and probably inhibited the ATPase. As follows from Fig. 2c,d, 5 · 10­5 M Cu2+ markedly lowered the maximal fluorescence yield (OP amplitude) while the JIP contribution and the curve shape changed less significantly. These results indicate a decrease of the number of functional PS II centers, possibly caused by suppressed repair [14]. The millisecond dark decay curves are resolved into three exponentials [23]. The fast and the medium components have been attributed to PS II reoxidation by the so called "fast" and "slow" PQ pools [29], though these kinetic fractions probably reflect the het

(F ­ Fo)/(FJ ­ Fo)

1.0


484

ANTAL et al.

Lifetimes () and amplitudes (A) of the fast (1), medium (2), and slow (3) components of the dark relaxation of variable fluorescence in C. moewusii cells incubated with MeHg and copper sulfate Exposure Control MeHg, 10­7 M CuSO4, 5 · 10­5 M Incubation, h 0 4 24 72 4 24 72 1, ms 22 8 9 18 4 ­ ­ 2, ms 75 33 37 42 62 66 52 3, ms 450 460 500 470 832 860 700 A1, % 58 27 24 29 24 0 0 A2, % 24 53 56 45 42 72 69 A3, % 18 20 20 26 34 28 31

Note: Decay measured with a PAM 2000 fluorimeter (see Experimental). Prior to measurements, the specimens were concentrated on a paper filter and kept in the dark for 5 min. Data averaged over four independently recorded kinetic curves were subjected to three exponential fitting.

erogeneity of PS II in the affinity for PQ, and/or dif ferences in PQ diffusion to and reoxidation at the cytochrome b6/f complex. The slow relaxation com ponent reflects recombination between Q A and the S2 state of the oxygen evolving complex in the centers with impaired electron transport to PQ [30]. Accord ingly, an increase in this component may indicate dis turbances in the PS II acceptor part. The table lists the results of three exponential anal ysis of the dark relaxation curves measured in the same incubations as above. In the control, the fast compo nent was dominant. Exposure to MeHg halved the amplitude of the fast component and correspondingly augmented the medium one, whereas the slow com ponent increased somewhat only in 72 h. Thus, MeHg caused the PS II oxidation rate to decrease in a sub stantial fraction of the ensembles (adding to the "slow PQ pool"), and made some more centers incapable of PQ reduction. The latter result corresponds to the above analysis of the OJIP transients (Fig. 2). The marked shortening of the lifetimes calculated for the fast and medium components in the presence of MeHg may be associated with partial inhibition of PQ reoxidation. Incubation with Cu2+ eliminated the fast compo nent within 24 h, at the same time tripling the share of the medium component (i.e., the slowly oxidized PS II fraction) and almost doubling both the amplitude and the lifetime of the slow component. The latter effect indicates accumulation of PS II centers that ­ cannot reduce PQ and have a lower rate of Q A ­S2 recombination [30], probably because of hindered S state transitions of the oxygen evolving complex, which is consistent with the data on the inhibitory action of Cu2+ on the PS II donor part [14, 15]. CONCLUSIONS The results presented here demonstrate the high sensitivity of PS II in the microalga C. moewusii to
­

copper ions and MeHg, the latter being toxic at much lower concentrations. Both agents can suppress elec tron transport at the acceptor side of PS II and increase the fraction of PS II corresponding to the "slow PQ pool." Besides, MeHg hinders PQ reoxida tion and increases the transmembrane pH gradient, which probably reflects impaired photophosphoryla tion in thylakoids. Analysis of the light induced transients and dark decay of chlorophyll fluorescence allows early detec tion of alterations caused by toxicants in algal cells, well before the onset of PS II destruction, and affords information on the possible mechanisms involved. This approach may prove useful in a variety of toxic and stress exposures. ACKNOWLEDGMENTS The work was supported by grant NSh 853.2008.4 and state contract 02.512.11.2213. REFERENCES
1. M. Bertrand and I. Poirier, Photosynthetica 43 (3), 345 (2005). 2. E. I. Ochiai, General Principles of Biochemistry of the Elements (Plenum Press, New York, 1987). 3. S. J. Stohs and D. Bagchi, Free Rad. Biol. Med. 18, 321 (1995). 4. W. E. Rauser, Cell Biochem. Biophys. 31, 1 (1999). 5. H. Luo, Y. Lu, X. Shi, et al., Ann. Clin. Lab. Sci. 26, 85 (1996). 6. V. Demidchik, A. Sololik, and V. Yurin, Plant Physiol. 114, 1313 (1997). 7. F. Navari Izzo and M. F. Quartacci, Minerva Bio tech. 13, 73 (2001). 8. P. Janeau, D. Dewez, S. Matsui, et al., Chemosphere 45, 589 (2001). 9. C. M. Lu, C. W. Chau, and J. H. Zhang, Chemosphere 41, 191 (2000).
BIOPHYSICS Vol. 54 No. 4 2009


ASSESSMENT OF THE EFFECTS OF METHYLMERCURY AND COPPER IONS 10. E. E. Graevskaya, T. K. Antal, D. N. Matorin, et al., J. Phys. IV France 107, 569 (2003). 11. T. K. Antal, E. E. Graevskaya, D. N. Matorin, et al., Biofizika 49 (1), 72 (2004). 12. G. P. Kukarskikh, E. E. Graevskaya, T. E. Krendeleva, et al., Biofizika 48 (5), 853 (2003). 13. A. Ahmed and H. A. Tajmirriahi, J. Inorg. Biochem. 50, 235 (1993). 14. D. V. Vavilin, V. A. Polynov, D. N. Matorin, and P. S. Venediktov, J. Plant. Physiol. 146, 609 (1995). 15. E. PÄtsikka, E. M. Aro, and E. TyystjÄrvi, Plant Phys iol. 117, 619 (1998). 16. A. B. Rubin, Tekhnol. Zhiv. Syst. 2, 47 (2005). 17. D. N. Matorin, T. K. Antal, M. Ostrowska, et al., Oceanologia 46, 519 (2004). 18. R. J. Strasser, M. Tsimilli Michael, and A. Srivastava, in Chlorophyll Fluorescence: a Signature of Photosynthe sis, Ed. by G.G. Papageorgiou and Govindjee (Kluwer Academic Publishers, Dordrecht Boston London, 2005), pp. 321­362. 19. U. Schreiber, J. Muller, A. Haugg, and R. Gademann, Photosynth. Res. 74, 317 (2002).

485

20. W. Brack and H. Frank, Ecotoxicol. Environ. Safety 40, 34 (1998). 21. D. V. Vavilin, J. M. Ducruet, D. N. Matorin, et al., J. Photochem. Photobiol. 42 (3), 233 (1998). 22. B. J. Strasser and R. J. Strasser, in Photosynthesis: from Light to Biosphere, Ed. by P. Mathis (Kluwer Academic Publishers, Dordrecht, 1995), vol. 4, pp. 909­912. 23. N. Bukhov, E. Egorova, T. Krendeleva, et al., Photo synth. Res. 70, 155 (2001). 24. T. K. Antal, T. Krendeleva, and A. Rubin, Photosynth. Res. 94, 13 (2007). 25. T. Antal and A. Rubin, Photosynth. Res. 96, 217 (2008). 26. D. LazÀr, Funct. Plant. Biol. 33, 9 (2006). 27. H. Dau and U. P. Hansen, Photosynth. Res. 25, 269 (1990). 28. A. A. Volgusheva, G. P. Kukarskikh, T. K. Antal, et al., Biofizika 53, 787 (2008). 29. P. Joliot, J. Lavergne, and D. Beal, Biochim. Biophys. Acta 1101, 1 (1992). 30. J. Cao and Govindjee, Biochim. Biophys. Acta 1015, 180 (1990).

BIOPHYSICS

Vol. 54

No. 4

2009