Photoinhibition of Chlamydomonas reinhardtii in State 1 and State 2
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m011376200
ISSN1083-351X
AutoresGiovanni Finazzi, Romina Paola Barbagallo, Elena Bergo, Roberto Barbato, Giorgio Forti,
Tópico(s)Algal biology and biofuel production
ResumoThe relationship between state transitions and photoinhibition has been studied in Chlamydomonas reinhardtii cells. In State 2, photosystem II activity was more inhibited by light than in State 1. In State 2, however, the D1 subunit was not degraded, whereas a substantial degradation was observed in State 1. These results suggest that photoinhibition occurs via the generation of an intermediate state in which photosystem II is inactive but the D1 protein is still intact. The accumulation of this state is enhanced in State 2, because in this State only cyclic photosynthetic electron transport is active, whereas there is no electron flow between photosystem II and the cytochromeb6 f complex (Finazzi, G., Furia, A., Barbagallo, R. P., and Forti, G. (1999) Biochim. Biophys. Acta 1413, 117–129). The activity of photosystem I and of cytochrome b6f as well as the coupling of thylakoid membranes was not affected by illumination under the same conditions. This allows repairing the damages to photosystem II thanks to cell capacity to maintain a high rate of ATP synthesis (via photosystem I-driven cyclic electron flow). This capacity might represent an important physiological tool in protecting the photosynthetic apparatus from excess of light as well as from other a-biotic stress conditions. The relationship between state transitions and photoinhibition has been studied in Chlamydomonas reinhardtii cells. In State 2, photosystem II activity was more inhibited by light than in State 1. In State 2, however, the D1 subunit was not degraded, whereas a substantial degradation was observed in State 1. These results suggest that photoinhibition occurs via the generation of an intermediate state in which photosystem II is inactive but the D1 protein is still intact. The accumulation of this state is enhanced in State 2, because in this State only cyclic photosynthetic electron transport is active, whereas there is no electron flow between photosystem II and the cytochromeb6 f complex (Finazzi, G., Furia, A., Barbagallo, R. P., and Forti, G. (1999) Biochim. Biophys. Acta 1413, 117–129). The activity of photosystem I and of cytochrome b6f as well as the coupling of thylakoid membranes was not affected by illumination under the same conditions. This allows repairing the damages to photosystem II thanks to cell capacity to maintain a high rate of ATP synthesis (via photosystem I-driven cyclic electron flow). This capacity might represent an important physiological tool in protecting the photosynthetic apparatus from excess of light as well as from other a-biotic stress conditions. photosystem 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea minimum value of fluorescence emission measured at open reaction centers maximal value of fluorescence emission measured at closed reaction centers variable fluorescence (Fm − Fo) singlet oxygen species plastocyanin plastoquinone plastoquinol primary quinone acceptor of photosystem II secondary quinone acceptor of photosystem II light-harvesting complex II The photochemical utilization of absorbed light is a critical step in the photosynthetic process. Because harvesting of light, photochemistry, and electron transfer occur on widely different scales of time, a correct balance among these different processes is required to optimize the efficiency of CO2 fixation. When light is absorbed in excess of what can actually be utilized by photochemistry, damage to the photosynthetic apparatus may be induced. Impairment of both photosystem I (PSI)1 (1Sonoike K. Plant Cell Physiol. 1995; 37: 239-247Crossref Scopus (224) Google Scholar) and photosystem II (PSII) (2Ohad I. Kyle D.J. Arntzen C.J. J. Cell Biol. 1984; 99: 481-485Crossref PubMed Scopus (371) Google Scholar) has been described, and this loss of activity has been termed photoinhibition (3Powles S.B. Annu. Rev. Plant Physiol. 1984; 35: 15-44Crossref Google Scholar). It has been also shown that the degradation of the PSII reaction center D1 subunit is a major consequence of photoinhibition (2Ohad I. Kyle D.J. Arntzen C.J. J. Cell Biol. 1984; 99: 481-485Crossref PubMed Scopus (371) Google Scholar). Some mechanisms contribute to protecting the photosynthetic apparatus from an excess of light (4Demmig-Adams B. Gilmore A.M. Adams W.W. FASEB J. 1996; 10: 403-412Crossref PubMed Scopus (605) Google Scholar, 5Niyogi K.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 333-359Crossref PubMed Scopus (1619) Google Scholar). The first is the so-called energy-dependent quenching, qE, i.e. the increased thermal dissipation in the PSII antennae that follows the generation of the electrochemical proton gradient across the thylakoid membranes. It is supposed to protect the reaction center from the consequences of a strong illumination by reducing the amount of energy present in the antenna protein complexes (6Horton P. Ruban A.V. Walters R.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 655-684Crossref PubMed Scopus (1421) Google Scholar). The second one (6Horton P. Ruban A.V. Walters R.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 655-684Crossref PubMed Scopus (1421) Google Scholar) is state transitions, a phenomenon that has been discovered in Chlorella pyrenoidosa (7Bonaventura C. Myers J. Biochim. Biophys. Acta. 1969; 189: 366-383Crossref PubMed Scopus (513) Google Scholar) and inPorphyridium cruentum (8Murata N. Biochim. Biophys. Acta. 1969; 172: 242-251Crossref PubMed Scopus (451) Google Scholar). It is a mechanism to balance light utilization between the two photosystems that is based on the reversible transfer of a fraction of the light-harvesting complex II (LHCII) from PSII to PSI (reviewed in Refs 9Bennett J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 281-311Crossref Scopus (234) Google Scholar, 10Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (730) Google Scholar, 11Gal A. Zer H. Ohad I. Physiol. Plant. 1997; 100: 869-885Crossref Google Scholar). It is also supposed to protect PSII from photoinhibition inasmuch as it can decrease the size of its antenna. The migration of LHCII to PSI (State 1-State 2 transition) results from the phosphorylation of the former by a membrane-bound protein kinase, which is activated under reducing conditions (reviewed in Refs. 9Bennett J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 281-311Crossref Scopus (234) Google Scholarand12). Under oxidizing conditions, the kinase is deactivated, and LHCII is dephosphorylated by a thylakoid-bound phosphatase, which is possibly regulated by the recently discovered immunophilin-like 40-kDa lumenal TLP protein (13Fulgosi H. Vener A.V. Altschmied L. Herrmann R.G. Andersson B. EMBO J. 1998; 17: 1577-1587Crossref PubMed Scopus (110) Google Scholar). After dephosphorylation, LHCII rebinds to PSII (State 2-State 1 transition). In higher plants, only a small fraction of the LHCII (15–20%, reviewed in Ref. 10Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (730) Google Scholar) migrates reversibly from PSII to PSI. In the green alga Chlamydomonas reinhardtii, on the contrary, a much larger fraction of the PSII antenna is transferred during State 1-State 2 transition (14Delosme R. Béal D. Joliot P. Biochim. Biophys. Acta. 1994; 1185: 56-64Crossref Scopus (47) Google Scholar), and a much larger decrease of PSII energy capture is accordingly observed (15Delosme R. Olive J. Wollman F.-A. Biochim. Biophys. Acta. 1996; 1273: 150-158Crossref Scopus (169) Google Scholar). In addition, cytochromeb6 f complexes accumulate in the unstacked lamellae in State 2 (16Vallon O. Bulté L. Dainese P. Olive J. Bassi R. Wollman F.-A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8262-8266Crossref PubMed Scopus (149) Google Scholar). Therefore, it is unlikely that state transitions serve the purpose of balancing the absorption of PSII and PSI in Chlamydomonas. Instead, State 2 would represent a structural condition where most of the excitation energy is utilized by PSI photochemistry so that cyclic electron transport around PSI is likely to prevail over linear electron flow that involves both PSI and PSII. In agreement with this idea, Finazzi et al. (17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar) show that although the cytochrome b6 f turnover was the same in State 1 and State 2, it was completely inhibited by the addition of the PSII inhibitor DCMU in State 1, whereas no effect of this inhibitor was observed in State 2. This result led Finazziet al. (17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar) to propose that in State 2 the reducing equivalents involved in the reduction of the cytochromeb6 f are not produced at the level of PSII but rather at the level of PSI. Under these conditions PSII is not connected to the intersystem electron carriers but is still photochemically active (17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar). To investigate whether this lack of functional connection between PSII and cytochrome b6 f complex might affect the sensitivity of the former to photoinhibition, we have measured the effects of strong illumination on fluorescence emission, O2 evolution, and cytochrome f reduction in algae under State 1 or State 2 conditions. We have found that PSII is more prone to photoinhibition in State 2. However, in this state the loss of activity is not accompanied by a degradation of the D1 protein. The effect on PSII seems to be rather specific as neither PSI nor cytochrome b6 f activities nor the coupling of thylakoid membranes were affected by the treatment. Thus, we suggest that state transitions in C. reinhardtiirepresent a means to maintain a high ATP synthesis capacity, even when damages to PSII are induced by illumination with extremely intense light. C. reinhardtiiwild type (from strain 137C) was kindly provided by the Laboratoire de Physiologie Membranaire du Chloroplaste at the Institut de Biologie Physico-Chimique of Paris (France). Cells were grown at 24 °C in acetate-supplemented medium (18Gorman D.S. Levine R.P. Proc. Natl. Acad. Sci. U. S. A. 1960; 46: 83-91Crossref PubMed Google Scholar) under 60 μE m−2 s−1 of continuous white light. They were harvested during exponential growth and resuspended at the required chlorophyll concentration in an high salt minimal medium (19Sueoka N. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1665-1669Crossref PubMed Scopus (1302) Google Scholar). The use of this medium prevented the spontaneous transition to State 2 otherwise observed in the presence of acetate (see Ref. 20Endo T. Asada K. Plant Cell Physiol. 1996; 37: 551-555Crossref Scopus (65) Google Scholar). 2F.-A. Wollman, personal communication.Chlorophyll concentration was measured as the absorbance at 680 nm of the cell cultures in a spectrophotometer equipped with a scatter attachment on the basis of a calibration curve constructed after extraction of the chlorophyll with 80% acetone. State 1 was obtained through incubation of the cells in the dark under strong agitation, whereas State 2 was obtained through dark incubation in anaerobic conditions obtained by argon bubbling. Photoinhibition was performed by illuminating the sample with white light on a thin layer (∼1 mm) of cells in a Petri dish ([chlorophyll] = 500 μg ml−1) at room temperature. The light was screened with a layer of water and infrared- and UV-absorbing filters. The intensity of the light reaching the sample was 2300 μE m−2 s−1. We ensured that the layer of cells was sufficiently thin to minimize mutual shadowing. When indicated, plastidial protein synthesis was inhibited by adding lincomycin at the final concentration of 1 mm. Samples were collected at the indicated times and used in the different experiments at the required chlorophyll concentration. Photosynthesis and respiration were measured as the O2 exchange with a Clark-type electrode (Radiometer, Denmark) at 24 °C. The actinic light was filtered through a heat filter, and its intensity was 850 μE m−2s−1. Fluorescence was measured in the same chamber used for O2 recordings using a PAM fluorometer (Walz, Germany). Spectroscopic measurements were performed on whole cells at room temperature using a homemade spectrophotometer as described by Joliot et al. (21Joliot P. Béal D. Frilley B. J. Chim. Phys. 1980; 77: 209-216Crossref Google Scholar). In continuous light experiments, actinic light was provided by a light-emitting diode array, placed on both sides of the cuvette. Its intensity was 1500 μE m−2s−1. Measurements were performed on algae kept under State 1 conditions obtained through a strong agitation in the dark in air. Estimation of the rates of cytochrome fturnover was done using a procedure previously employed (17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar). Briefly, starting from the consideration that the rate of cytochromef oxidation and reduction is the same at steady state, the latter can be expressed as df −/dt = [f−] × kox × [PC+], where [f−] is the fraction of reduced cytochrome f, and kox × [PC+] represents the product of the second order rate constant for cytochrome foxidation times the concentration of oxidized plastocyanin. Both parameters can be easily calculated experimentally from the traces of Fig. 3; [f−] is estimated comparing the plateau absorption level measured in the absence and presence of DBMIB, whereaskox × [PC+] is given by the initial rate of cytochrome f oxidation provided that it is measured when its reduction is inhibited, i.e. in the presence of DBMIB. In single turnover flash experiments, excitation was provided by a xenon lamp (EG&G). Light was filtered through a Schott filter (RG 695) and was of saturating intensity. Measurements were performed on algae kept in State 2 to ensure dark reduction of the plastoquinone pool. Repetitive (usually 10) illuminations were performed at the frequency of 0.15 Hz. The transmembrane potential was estimated from the amplitude of the electrochromic shift at 515 nm, which is known to give a linear response with respect to the electric component of the transmembrane potential (22Junge W. Witt H.T. Z. Naturforsch. 1968; 24: 1038-1041Google Scholar). Under the conditions employed here, the kinetics of the electrochromic signal exhibited two phases previously characterized in Joliot and Delosme (23Joliot P. Delosme R. Biochim. Biophys. Acta. 1974; 357: 267-284Crossref PubMed Scopus (160) Google Scholar): a fast phase (phase a), associated with PSI and PSII charge separation, and a slow phase, which develops in the millisecond time scale and is associated with the turnover of the cytochrome b6 fcomplex (phase b). The kinetics of phase b was deconvoluted from membrane potential decay assuming that the latter process exhibited first-order kinetics. Phase b was then computed considering that the rate of membrane potential decay between two consecutive acquisitions was linearly related to its mean value in the same interval. Cytochrome f redox changes were evaluated as the difference between the absorption at 554 nm and a base line drawn between 545 and 573 nm. We have checked that this procedure for deconvolution of cytochrome f signals was reliable also in the case of continuous illumination (17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar). For protein analysis, algae were collected at the indicated times, washed in 20 mm HEPES containing protease inhibitors (200 μm phenylmethylsulfonyl fluoride, 5 mm amino-ε-caproic acid, and 1 mmbenzamidine) and 1 mm lincomycin, and resuspended in 100 mm dithiothreitol, 100 mmNa2CO3. The algae were then solubilized in the presence of 2% SDS and 20% (w/v) sucrose at 100 °C for 1 min. Polypeptides were separated by denaturing SDS-polyacrylamide gel electrophoresis in the presence of 6 m urea. Immunoblotting was performed with monospecific polyclonal antibodies against D1, as described in Barbato et al. (24Barbato R. Shipton C.A. Giacometti G.M. Barber J. FEBS Lett. 1991; 290: 162-166Crossref PubMed Scopus (79) Google Scholar). To investigate the influence of state transitions on the sensitivity of C. reinhardtii to photoinhibition, we have performed experiments on cells placed either in State 1 or in State 2. We have measured fluorescence emission, photosynthetic activity (as O2 evolution), and the rate of cytochrome freduction after exposure to strong illumination. The measurements were performed on the same batch of algae, collected either before starting the light treatment or after different irradiation times. Illumination of the algae with high light intensity largely modified their fluorescence emission parameters; a large decrease of the maximal fluorescence emission (Fm) was observed in State 1 cells (Fig.1 A), whereas an increase of the minimal one (Fo) occurred in State 2 (Fig. 1 B). This suggests that the consequences of illumination on the photosynthetic apparatus of Chlamydomonas were not identical in the two conditions. In both cases, however, the effect of illumination was to reduce the Fv/Fm ratio (Fig. 1 C), a parameter related to the photochemical efficiency of PSII (25Butler W.L. Ann. Rev. Plant Physiol. 1978; 29: 345-378Crossref Google Scholar). This indicates that PSII was the major target of photoinhibition in both State 1 and State 2. The Fv/Fm decline was reversible in the dark, unless an inhibitor of protein synthesis, lincomycin, was present in the medium (not shown). The addition of this compound during illumination enhanced the photoinhibition, and its effect was larger in State 2 (Fig.1 D). Samples were also collected to measure the effects of illumination on the photosynthetic O2 evolution. To this aim, State 1 was re-established (by oxygenation in the dark) in algae preilluminated in State 2 before O2 evolution was recorded. We have already shown indeed that no oxygen is evolved by the algae in State 2 (17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar). During the State 2 to State 1 transition, no recovery of inhibition occurred; after the transition to State 1, the Fv/Fm of State 2-treated algae was still largely inhibited if compared with an untreated (State 1) sample (not shown). The oxygen evolution rates (measured before and after the photoinhibitory treatment) are shown in TableI. No decrease of oxygen evolution was observed in both State 1- and State 2-illuminated samples in the absence of lincomycin. In its presence, a loss of activity was observed, which was again larger in State 2- than in State 1-treated cells.Table IEffects of photoinhibition on photosynthetic oxygen evolution in Chlamydomonas cellsTimeState 1State 2ControlLincomycin, 1 mmControlLincomycin, 1 mmmin0105 ± 10104 ± 598 ± 10112 ± 1020110 ± 8106 ± 793 ± 1125 ± 1130109 ± 11100 ± 1097 ± 1113.5 ± 860111 ± 983 ± 8100 ± 99 ± 690108 ± 1054 ± 699 ± 79 ± 9Oxygen evolution and consumption were measured by a Clark-type electrode at a chlorophyll concentration of 20 μg ml−1 in the presence of 5 mm NaHCO3. Photosynthetic oxygen evolution rate (μmol mg−1 chlorophyll h−1) is expressed as the sum of O2 evolution (in the light) and consumption (in the dark). The latter was not affected by the treatment. Other conditions are as in Fig. 1. Light intensity was 850 μE m−2 s−1. Data represent the result of five different experiments. Open table in a new tab Oxygen evolution and consumption were measured by a Clark-type electrode at a chlorophyll concentration of 20 μg ml−1 in the presence of 5 mm NaHCO3. Photosynthetic oxygen evolution rate (μmol mg−1 chlorophyll h−1) is expressed as the sum of O2 evolution (in the light) and consumption (in the dark). The latter was not affected by the treatment. Other conditions are as in Fig. 1. Light intensity was 850 μE m−2 s−1. Data represent the result of five different experiments. A more direct way to characterize the effects of irradiation on PSII photochemical activity would be to measure directly the rate of plastoquinone reduction. It is very difficult to measure this parameter in vivo, where the redox changes associated to PQH2 formation (observed around 260 nm) are largely masked by other absorption signals. However, it is possible to obtain this information indirectly by measuring the rate of cytochromef reduction. This rate can be expressed indeed as kred × [f·Fe3+S·bl+ ·bh] × [PQH2], where kred is the second order rate constant for plastoquinol oxidation, [f·Fe3+S·bl+ ·bh] represents the concentration of active cytochromeb6 f complexes, and [PQH2] expresses the concentration of plastoquinol. Although [PQH2] is proportional to the fraction of active PSII, at least in State 1 conditions (Ref. 17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar, see also below), the product kred × [f·Fe3+S·bl+ ·bh] depends on the catalytic efficiency of the cytochrome complex. Therefore, it is important first to check that the turnover rate of cytochrome b6 f complex per se is not affected by the photoinhibitory treatment. Only in this case, the product kred × [f·Fe3+S·bl+ ·bh] can be taken as a constant, and the cytochrome f reduction rate can be used to obtain information on PSII activity. The intrinsic cytochrome b6 f activity can be easily measured in State 2 conditions (i.e.anaerobiosis) under a single turnover flash illumination regime of low actinic light frequency. In these conditions indeed the PQ pool is fully rereduced in the dark time between two consecutive illuminations (26Finazzi G. Buschlen S. de Vitry C. Rappaport F. Joliot P. Wollman F.-A. Biochemistry. 1997; 36: 2867-2874Crossref PubMed Scopus (39) Google Scholar), and the catalytic properties of the complex can be studied independently of the rate of PQ photoreduction. Thus, the kinetics of cytochrome b6f under single flash illumination was always measured in State 2: State 1 preilluminated cells were dark-adapted to anaerobiosis before their cytochromeb6 f kinetics were measured. Fig. 2 shows the kinetics of the electrochromic shift (panels A and B) and of cytochrome f redox changes (panel C andD) measured before and after a photoinhibitory treatment ofC. reinhardtii. The slow phase of the electrochromic shift (phase b, see "Materials and Methods") and cytochrome fredox changes are representative of electron injection into the low and high potential electron transfer chains of the cytochromeb6 f complex, respectively (23Joliot P. Delosme R. Biochim. Biophys. Acta. 1974; 357: 267-284Crossref PubMed Scopus (160) Google Scholar, 26Finazzi G. Buschlen S. de Vitry C. Rappaport F. Joliot P. Wollman F.-A. Biochemistry. 1997; 36: 2867-2874Crossref PubMed Scopus (39) Google Scholar). Fig. 2 shows that all the electron transfer steps that follow plastoquinol oxidation were not affected by the photoinhibitory illumination. No differences were observed between cells treated in State 1 and State 2 (not shown). During the measurements, PSII activity was inhibited with DCMU and hydroxylamine (27Bennoun P. Biochim. Biophys. Acta. 1970; 216: 357-363Crossref PubMed Scopus (206) Google Scholar). Their addition did not affect cytochromeb6 f kinetics (as expected, since it does not depend on PSII activity in State 2, see above) but reduced the amplitude of the fast phase of the electrochromic signal (phase a) because of the loss of PSII photochemistry. In the presence of DCMU and hydroxylamine, phase a only depends on PSI-driven charge separation. Its constancy before and after the photoinhibitory treatment (Fig. 2, A and B) suggests that PSI was not affected by photoinhibition in our conditions. The treatment did not affect the permeability of the thylakoid membrane either, as indicated by the finding that the addition of a ionophore induced the same acceleration of cytochromeb6 f turnover in both untreated and treated cells. The kinetic effect of ionophores is quantitatively related to the magnitude of the electrochemical proton gradient (reviewed in Ref. 28Bendall D. Biochim. Biophys. Acta. 1982; 683: 119-151Crossref Scopus (121) Google Scholar), i.e. to the permeability of membrane to ions. This conclusion is in agreement with previous results with isolated thylakoid membranes (29Barenyi B. Krause G.H. Planta. 1985; 163: 218-226Crossref PubMed Scopus (104) Google Scholar, 30Forti G. Barbagallo R.P. Inversini B. Photosynth. Res. 1999; 59: 215-222Crossref Google Scholar). Having ascertained that the intrinsic activity of PSI and cytochromeb6 f complex was not affected by photoinhibition, we have estimated the consequences of illumination on PSII by measuring the turnover rate of cytochrome f under steady state illumination conditions, as stated above. Fig. 3 shows the results of such measurements in the case of one representative experiment. Panels A and B refer to measurements performed on dark-adapted algae under both State 1 (A) and State 2 (B). Similar cytochrome f kinetics was observed in both states; switching the light on generated an oxidation signal (absorption decrease) that rapidly attained a plateau level. After the light was switched off, a reduction was observed that brought the signal to its initial level. In State 1 and State 2 conditions, the extent of the oxidation signal was equally sensitive to the addition of DBMIB, an inhibitor of cytochrome f reduction by plastoquinol (31Frank K. Trebst A. Photochem. Photobiol. 1995; 61: 2-9Crossref PubMed Scopus (28) Google Scholar) (Figs. 3, A and B, compare squares andtriangles). DCMU, which blocks plastoquinone reduction by PSII (27Bennoun P. Biochim. Biophys. Acta. 1970; 216: 357-363Crossref PubMed Scopus (206) Google Scholar), inhibited electron flow only in State 1 cells (circles). This result confirms previous findings from Finazzi et al. (17Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (139) Google Scholar) that the transition from State 1 to State 2 corresponds to a shift from a linear (involving PSII and PSI) to a cyclic (involving only PSI) electron transport system. Therefore, in State 2 it is not possible to measure PSII activity on the basis of cytochrome f turnover. For this reason, the consequences of preillumination on cytochrome f kinetics in the case of continuous illumination regime were measured in State 1, at variance with single flash measurements. In State 2-preilluminated cells, State 1 was re-established by dark oxygenation of the cells. The same treatment did not affect the rate of electron transfer in dark-adapted cells (not shown). Panels C and D of Fig. 3 present the results of such measurements. In the absence of lincomycin, no differences were observed between preilluminated and dark-adapted cells (squares). The steady state redox level of cytochrome f was more oxidized, however, in lincomycin-treated samples (asterisks). This suggests that photoinhibition reduced the rate of plastoquinol generation by PSII in the presence of the antibiotic, in agreement with the finding that O2 evolution was inhibited (Table I). This conclusion is also in agreement with previous results obtained under similar experimental conditions in higher plant leaves (32Chow W.S. Hope A.B. Aust. J. Plant Physiol. 1998; 25: 775-784Crossref Scopus (22) Google Scholar). Again, the consequences were more severe in the case of State 2- than State 1-treated cells (compare C and D, asterisks). In untreated cells, lincomycin did not affect cytochrome f turnover (not shown). The time courses of the decrease in PSII-driven cytochrome f electron flow are shown in TableII.Table IIEffect of photoinhibition on the electron flow through cytochrome b6fTimeState 1State 2ControlLincomycin, 1 mmControlLincomycin, 1 mmmin070 ± 565 ± 768 ± 574 ± 52065 ± 656 ± 767 ± 618 ± 33079 ± 748 ± 362 ± 49 ± 46071 ± 735 ± 465 ± 56 ± 49066 ± 826 ± 570 ± 63 ± 3The rates of cytochrome f reduction were calculated from traces as in Fig. 3 as explained under "Materials and Methods". Rates are expressed as mol e− s−1. Data represent the result of five independent experiments. Open table in a new tab The rates of cytochrome f reduction were calculated from traces as in Fig. 3 as explained under "Materials and Methods". Rates are expressed as mol e− s−1. Data represent the result of five independent experiments. All the measurements performed so far indicate that the activity of PSII is decreased by photoinhibition both in State 1 and State 2 provided that lincomycin is added to the cell suspension. This loss of activity is generally associated to a damage of the D1 subunit of PSII, which is subsequently rapidly degraded (see e.g. Refs. 2Ohad I. Kyle D.J. Arntzen C.J. J. Cell Biol. 1984; 99: 481-485Crossref PubMed Scopus (371) Google Scholar and 33Kyle D.J. Ohad I. Arntzen C.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4070-4074Crossref PubMed Google Scholar). To verify if this was the case in our conditions, we have measured the amount of D1 in both State 1- and State 2-treated samples using an immunoblotting essay (Fig. 4). We found remarkable differences between State 1 and State 2 cells; although the amount of D1 was reduced upon photoinhibition in State 1-treated cells, no substantial degradation was observed in State 2 despite a massive loss of PSII activity (Fig. 4 A). It has previously been demonstrated that DCMU protects the D1 protein from degradation (34Trebst A. Depka B. Kraft B. Johanningm
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