Pa.ta
Planta 9 Springer-Verlag 1994
The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage Jacques Ravenei, Gilles Peltier, Michel Havaux D~partement de PhysiologieV~g6taleet Ecosyst~mes,CEA-Sciencesdu Vivant, Centre d'Etudes de Cadarache, F-13108 Saint-Paul-lez-Durance,France Received: 22 July 1993/ Accepted: 2 November 1993
Abstract. The photoacoustic technique was used to measure energy storage by cyclic electron transfer around photosystem I in intact Chlamydomonas reinhardtii cells illuminated with far-red light (>715 nm). The in-vivo cyclic pathway was characterized by investigating the effects of various chemicals on energy storage. Participation of plastoquinone and ferredoxin in the cyclic electron flow was confirmed by the complete suppression of energy storage in the presence of the plastoquinol antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the ferredoxin inhibitors/competitors methylviologen, phenylmercuric acetate and p-benzoquinone. Two alternative electron cycles are demonstrated to operate in vivo. One cycle is sensitive to antimycin A, myxothiazol and 2-(n-heptyl)-4-hydroxyquinoline Noxide (HQNO) and is catalyzed by ferredoxin which reduces plastoquinone through a route involving cytochrome b 6 and its protonmotive Q-cycle. The other cycle is unaffected by the above-mentioned inhibitors but is sensitive to N-ethylmaleimide (NEM), an inhibitor of the ferredoxin-NADP reductase, and 2'-monophosphoadenosine-5'-diphosphoribose (PADR), an analogue of NADP, showing that the electron recycling was mediated by NADPH. Possibly, electrons enter the plastoquinone pool through the action of a NAD(P)H dehydrogenase, which is insensitive to classical inhibitors of the mitochondrial NADH dehydrogenase. Loss of energy storage by photosystem-I-driven cyclic electron transfer in farred light was observed only when antimycin A, myxothiazol or HQNO was used in combination with NEM or PADR. Analysis of the light-intensity dependence and Abbreviations: AmaX=maximalphotothermal signal; Cyt=cytochrome; DBMIB= 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU (diuron)=3-(3,4-dichlorophenyl)-l,l-dimethylurea; ES =photochemical energy storage; FNR= ferredoxin NADP+ reductase; HQNO=2-(n-heptyl)-4-hydroxyquinoline N-oxide; NEM=N-ethylmaleimide; P7oo=reaction-center pigment of PSI; PADR=2'-monophosphoadenosine-5'-diphosphoribose; pBQ= p-benzoquinone; PMA =phenylmercuricacetate Correspondence to: M. Havaux; FAX: (33)42254656
the rate of in-vivo cyclic electron transfer in the presence of various inhibitors indicates that the NADPH-dependent electron-cycle is the preferential cyclic pathway in Chlamydomonas cells illuminated with far-red light.
Key words: Chlamydomonas - Cyclic electron transport Photoacoustics - Photosystem I
Introduction In oxygenic photosynthetic organisms, PSI and PSII function in series to drive a "linear" electron flow from H20 to NADP + with the resultant evolution of molecular oxygen and the concomitant synthesis of ATP. Besides this linear electron transfer, both PSI and PSII can work independently in a cyclic electron transport. During PSI-driven cyclic electron transfer, a proton gradient is generated for the synthesis of ATP with no net production of NADPH. It has been suggested that the main role of cyclic electron flow through PSI is to adjust the ratio of ATP to NADPH to the value required for carbon assimilation (Schfirmann et al. 1971; Chain and Arnon 1977; Heber et al. 1978; Hind et al. 1981). Additionally, ATP-producing cyclic flow around PSI could play a role in some stress situations such as strong light stress when PSII is selectively damaged (Canaani et al. 1989) or salinity stress when extra ATP is required for active ion transport (Jeanjean et al. 1993). Membrane energization by PSI cyclic activity could also be involved in the modulation of the non-photochemical energy dissipation in PSII, e.g. during water stress when CO2 is not freely available (Katona et al. 1992). One of the remaining question about PSI cyclic electron flow pertains to the electron-transport component involved. The current perception is that cyclic and linear pathways share a common sequence of carriers, with the cyclic electron flow branching at ferredoxin and reentering between plastoquinone and plastocyanin (Bendall 1982; Crofts and Wraight 1983). Presumably, the par-
252
J. Ravenel et al. : The in-vivo cyclic electron pathways around PSI
titioning o f electrons between cyclic and noncyclic pathways at the level o f ferredoxin is regulated by the N A D P H / N A D P + ratio (Slovacek et al. 1980; Hosler and Y o c u m 1985, 1987). There are reports suggesting that the cyclic p a t h w a y also includes f e r r e d o x i n - N A D P + reductase ( F N R ) but it is n o t clear whether this enzyme functions as an integral c o m p o n e n t o f the p a t h w a y (Hind et al. 1981 ; S h a h a k et al. 1981) or plays a regulatory role (Cleland and Bendall 1992). Neither the m e c h a n i s m by which reducing equivalents return to p l a s t o q u i n o n e nor the involvement o f c y t o c h r o m e (Cyt) b 6 in this step is clear. Further, the complexity o f the cyclic PSI activity with the possible existence o f different electron routes is illustrated by the fact that the degree o f inhibition o f cyclic p h o t o p h o s p h o r y l a t i o n o f thylakoid fragments by antimycin A substantially varies with the redox conditions (Hosler and Y o c u m 1987). M o s t o f the previous studies o f the PSI-driven cyclic electron flow and the associated p h o s p h o r y l a t i o n were p e r f o r m e d on thylakoid p r e p a r a t i o n s supplemented with artificial cofactors or with exogenous ferredoxin so that the representativeness o f those artificial systems to the in-vivo situation can be questioned. O n the other hand, in vivo analyses o f cyclic electron flow in whole cells or tissues used light-induced a b s o r b a n c e changes which are indirectly related to the cyclic PSI activity (e.g. L e e g o o d et al. 1983). The technique o f p h o t o a c o u s t i c s provides a new w a y to p e r f o r m in vivo quantitative measurements o f the t h e r m o d y n a m i c efficiency o f cyclic PSI electron flow (for a review, see F o r k and H e r b e r t 1993). This convenient a p p r o a c h has been successfully applied to the detection o f cyclic electron-transport activity in various plant materials f r o m c y a n o b a c t e r i a to higher-plant leaves ( C a n a a n i et al. 1989; H e r b e r t et al. 1990; H a v a u x 1992; Jeanjean et al. 1993). In this study, we have employed the p h o t o a c o u s t i c m e t h o d to study the effects o f various chemicals on the cyclic PSI activity in Chlamydomonas cells, with the aim o f characterizing the in-vivo cyclic electron pathway(s). Near-infrared absorbance measurements o f the oxidation-reduction state o f Pv0o (the reaction center o f PSI) were also performed, providing an additional s u p p o r t to o u r interpretation o f the p h o t o a c o u s t i c data. The presented results reveal the existence o f two alternative cyclic p a t h w a y s in vivo: one which is catalyzed by ferredoxin and the other mediated by N A D P H .
diphosphoribose (PADR) in the algae, an electroporation technique was used (Brown et al. 1991). To this end, the algal cells, suspended in a 2.5 mM Tris buffer containing 10 mM KC1 and the chemicals under investigation (see list in Table 3), were subjected to two consecutive 20-ms pulses of high-voltage electric field (2000V-cm -1) delivered at intervals of 10s using a Jouan GHT1287B electropulsator (Jouan, Saint-Herblain, France). After incubation/electroporation, the algal suspension was filtered under pressure through an MF-Millipore filter (cellulose nitrate/acetate, SS type, 3 ~tm pore size; Millipore, Bedford, Mass., USA). The algae deposited on the filter (diameter, 1.2 cm) were then placed in the photoacoustic cell for measurements.
Material and methods Algae. Chlamydomonas reinhardtii Dang. (wild type, type 2137) was grown phototrophically at 20~ as described by Peltier and Thibault (1983). The mutant strains F15 + and F34, deficient in PSI and PSII, respectively, were grown in dim light in a Trisacetate-phosphate medium as described elsewhere (Bennoun 1982). The algae were generously provided by Dr. P. Bennoun (Institut de Biologic Physico-chimique, Paris, France). Treatments. After low-speed centrifugation (1500'9), the algae were resuspended and incubated for around 30 min in a buffer of pH 7.2 (50 mM Tris, 0.2 mM KC1) to which specific chemicals (listed in Tables 1, 2 and 4) were added. For the introduction of NADP and the NADP-analogue 2'-monophosphoadenosine-5'-
Photoacoustic measurements of photochemical energy storage. Energy storage (ES) by cyclic electron flow around PSI was measured in vivo using the photoacoustic technique, as described in detail elsewhere (Canaani et al. 1989; Herbert et al. 1990; Havaux 1992). In brief, the photoacoustic method quantifies the conversion of light energy to heat in a sample (Rosencwaig 1980) and hence, by comparison to a reference (light-saturated) state where the conversion is maximal, the storage of light energy as chemical energy (Bults et al. 1982). When monitored in far-red light (> 700 nm) chiefly absorbed in PSI, ES is specifically related to the PSI function, reflecting energy storage in photochemical products associated with the cycling of electrons around PSI (Canaani et al. 1989; Herbert et al. 1990). Excitation of PSII by the far-red light was insignificant since no 02 evolution could be detected and no effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the ES value was observed (cf. Results and Discussion). In practice, a sample of plant material, placed in a hermetically closed cell, is illuminated with intensity-modulated light, causing the generation of periodic pulses of heat. Due to the periodic expansion and contraction of a thin layer of gas adherent to the sample, an acoustic wave is created in the bulk gas phase, which propagates in the photoacoustic cell where it is detected by a sensitive microphone. The amplitude (A) of the photoacoustic signal is then proportional to the thermal deactivation of excited pigment molecules. When a strong, photosynthetically saturating, nonmodulated light is added to the modulated measuring light, the absorbed modulated light energy is almost completely dissipated as heat, yielding the maximal photothermal signal (amplitude AmaX)which is thus proportional to the light energy absorption of the sample. Neglecting the low-yield chlorophyll fluorescence emission, the extent of the photochemical energy storage can be calculated as ES = (A. . . . A)/Am"x(Bults et al. 1982). The photoacoustically monitored ES is a measure of the efficiency of photochemistry comparable to the quantum yield. The extent of the photoacoustically measured ES depends, however, on the modulation frequency of the exciting light (Malkin and Cahen 1979). Indeed, energy storage during one cycle is due to intermediates that did not decay during this cycle and, consequently, ES is expected to increase at higher frequencies due to storage of higher energies in intermediates closer to the primary photoact. More precisely, ES senses photochemical products with a lifetime higher than the reciprocal of the angular frequency (Malkin and Cahen 1979), i.e. approx. 9 ms at the modulation frequency used in this study (17 Hz). In view of this rather long lifetime, ES by cyclic PSI activity has been hypothesized to represent photophosphorylation of ADP (Herbert et al. 1990), reduction of plastoquinone molecules (Carpentier et al. 1990) or changes in the reduction-oxidation state of ferredoxin and Cyt b6/fcomplex (Cha and Mauzerall 1992). The experimental results presented below are compatible with these views and suggest NADPH as another energy-storing molecules involved in the measured ES. Light from a 1000-W quartz tungsten halogen lamp (Oriel 6317 ; Oriel, Stratford, Conn., USA) was filtered through a combination of filters (Oriel 6213 water filter, UV-absorbing Oriel KV 418 filter and RG715 cut-off filter; Schott, Mainz, Germany) and was chopped at 17 Hz using a rotating-wheel chopper (Model 218; Bentham Instruments, Reading, UK). The fluence rate of the resulting far-red light (maximal value 90 W - m-2) was adjusted using Schott neutral density filters. When the RG715 filter was replaced by a blue-green
J. Ravenel et al. : The in-vivo cyclic electron pathways around PSI BG38 filter (Schott), the measured ES reflected photochemical events involving both, photosystems (i.e. mainly linear electron flow). In order to avoid interference of modulated 02 evolution, ES measurements in blue-green light were performed at high frequency (297 Hz) where the photoacoustic signal is purely photothermal (Bults et al. 1982). One branch of a randomized polyfurcated light guide (101-F; Walz, Effeltrich, Germany) was used to pass the modulated light to the sample placed in a photoacoustic cell similar to that described by Cahen (1981). Another arm was used to superimpose (when needed) a strong non-modulated saturating white light (175W "m -z) produced by a Schott KL1500 light source. The photoacoustic signal detected by the microphone (BL-1785; Knowles, Franklin Park, III., USA) was processed by a lock-in amplifier (SR530; Stanford Research Systems, Sunnyvale, Cal., USA) and was displayed on a chart recorder. Photoacoustic measurements are performed in a tiny closed cell and, therefore, the exact 02 and CO2 concentrations are unknown. However, we can exclude that the samples became anaerobic by dark respiration in the small cell. Indeed, anaerobiosis is known to induce reduction of the plastoquinone pool (Diner and Mauzerall 1973), and chlorophyll fluorescence measurements (not shown in this study) have clearly demonstrated that this was not the case under our experimental conditions. Redox state of P700. Changes in the redox state of the reaction
center Pv0o of PSI were monitored via cell absorbance changes at around 830 nm (Inoue et al. 1973) using an ED-800-T emitter/ detector unit (Walz) connected to a Walz PAM-101 system (Schreiber et al. 1988). Short pulses (1 gs) of measuring light were obtained from a 830-nm light-emitting diode covered with a Schott RG780 cut-off filter and was applied repetitively at 100 kHz (integrated fluence rate 0.9 W - m - Z ) . The reaction center Pvoo was oxidized by illuminating the algae with "strong" far-red light (730 nm, 15 W 9m -2) supplied by a Schott KL1500-E light source and an interference filter (S10-730-F; Corion, Holliston, Mass., USA). The kinetics of P+oo re-reduction in the dark after interrupting the far-red light was recorded with a storage oscilloscope (5111A; Tektronix, Guernsey, Channel Islands). The half-time of the P4-ooreduction in the dark was used as a measure of the turnover of Pv00 in the cyclic pathway (Maxwell and Biggins 1976). A MACAM Q101 radiometer (MACAM Photometrics, Livingston, UK) was used to measure light fluence rates.
Mitochondrial respiration. The rate of 02 consumption by Chlamydomonas cells in the dark and its reduction by various inhibitors of the mitochondrial NADH-ubiquinone oxidoreductase (rotenone, 9(10 H)-acridone, amobarbytal and 4-hydroxypyridine) were measured with a Clark-type 02 electrode (DW2/2 ; Hansatech, King's Lynn, UK) as described by Delieu and Walker (1972).
253
~--A 1 rain I
13-
10 o) 5
J
0
Figure 1 (trace 1) shows the p h o t o t h e r m a l signal generated by Chlamydomonas cells deposited on a nitrocellulose filter and illuminated with a modulated farred light ( > 715 nm) in the presence or absence of a strong, photosynthetically saturating, continuous white light. In the presence of the saturating background light, the absorbed (modulated) light energy is almost completely dissipated as heat, causing a marked rise in the signal amplitude. The comparison of the actual photothermal signal (A) and the light-saturated reference level (A max) provides a direct estimate of the a m o u n t of absorbed far-red light energy which was stored by the photochemical processes (Bults et al. 1982; for detail see Materials and methods). The heat-emission signal shown in Fig. 1 was obtained with a light fluence rate (approx.
.-I
Fig. 1. Trace 1, photoacoustic signal (arbitrary units) generated by filter-deposited Chlamydomonas reinhardtii cells (wild type) in wavelengths of measuring light predominantly absorbed in PSI A (>715nm; 17Hz; 4 0 W - m - 2 ; ~, on; 5 ' Off). Photochemistry was transiently saturated (at the time indicated by the upward-pointing arrow ~ ) by a strong background light (175 W 9m-2), resulting in the maximal dissipation of absorbed light energy as heat. Comparison of the maximal signal amplitude (Amax)with the amplitude (A) of the actual photothermal signal allows ES to be estimated as: ES = (Am~X-A)/A max. I1,, background saturating light off. Time constant of the lock-in amplifier was 300 ms. Traces 2, 3 show the effects of the saturating background light on the photothermal signal of the F15 § and F34 mutant strains, respectively. The ES of the F34 mutant strain (Trace 3) was 5%
0
Results
|
20 40 Fluence rate of
60
the modulated far-red light ON m -a)
Fig. 2. Plot of the reciprocal of ES versus the fluence rate of the measuring far-red light (> 715 nm, 17 Hz) in Chlamydomonas cells
40 W 9m - 2 ) in the actinic range which can cause partial saturation of the electron-transport reactions, thus decreasing the apparent efficiency of photochemical energy storage (ES). This is clearly shown in Fig. 2 where the effects of the ttuence rate o f the modulated far-light on the magnitude of ES was examined. The plot, which was linearized by plotting the reciprocal of ES versus the light fluence rate (Havaux et al. 1989), showed a m a r k e d effect of the far-red light fluence rate on ES. The extrapolation of 1/ES to a modulated light fluence rate of zero gives an estimation of the maximal efficiency of photochemical
254
J. Ravenel et al. : The in-vivo cyclic electron pathways around PSI
energy storage; this extrapolated value of ES will be used in the following experiments. In the example of Fig. 2, approx. 35% of the absorbed far-red light was stored in photochemical products; this is close to values previously measured in other algal species (Herbert et al. 1990; Cha and Mauzerall 1992). In our experiments, the maximal ES of Chlamydomonas cells was found, however, to vary with the physiological state o f the algae, ranging from about 20% to 40%. As far-red light ( > 7 1 5 nm) is almost exclusively absorbed by PSI (Greenbaum and Mauzerall 1991), the measured ES has been related to cyclic electron flow around PSI (Canaani et al. 1989; Herbert et al. 1990). The participation of PSI was confirmed by two independent observations: (i) ES in far-red light was detected in a mutant strain (F34) of Chlamydomonas deficient in PSII but not in a mutant strain (F15 +) lacking functional PSI (Fig. 1, traces 2, 3) and (ii) ES in far-red light is insensitive to D C M U , a well-known inhibitor of electron transfer at the reducing side of PSII (Table 1 ; see also Canaani et al. 1989; Herbert et al. 1990). Note that, in conditions where the linear electron-transport system was completely blocked by 10 ~tM D C M U , as indicated by separate O2-evolution measurements (data not shown), an appreciable ES of approx. 20 % of the control value (measured before D C M U poisoning) was still observed in broadband modulated light (297 Hz). This remaining activity can be attributed to cyclic PSI activity in blue-green light. Table 1 shows the effects of various chemicals on the maximal efficiency of cyclic electron transport around PSI in Chlamydomonas cells as measured by ES. The participation of plastoquinone to the PSI-driven cyclic electron transfer is confirmed by the strong inhibitory effect of 2,5-dibromo-3-methyl-6-isopropyl-P-benzoquinone (DBMIB; see also Canaani et al. 1989; Herbert
et al. 1990) which is an inhibitor of the Cyt b6/fcomplex blocking the binding of plastoquinol to the oxidizing Qz site (Hauska et al. 1983). P-Benzoquinone (pBQ) and methylviologen which accept electrons at the acceptor side of PSI (Trebst 1974), thus competing with ferredoxin, were observed to strongly inhibit PSI-cyclic electron transport whereas ES by linear electron transport in blue-green light was significantly less affected. Phenylmercuric acetate (PMA) also interacts with the acceptor side of PSI by selectively inhibiting ferredoxin at the concentration used here (40 ~tM) (Honeycutt and Krogmann 1972), resulting in a loss of ES in both far-red and blue-green light beams. The observed effects of PMA, pBQ and methylviologen confirm the participation of ferredoxin to the electron cycling around PSI in vivo. Antimycin A is a well-known inhibitor of ferredoxincatalyzed cyclic electron transport around PSI in vitro (Tagawa et al. 1963). Surprisingly, this compound did not affect the cyclic electron flow in vivo. In the same way, the cytochrome inhibitors 2(N-heptyl)-4-hydroquinoline N-oxide (HQNO) and myxothiazol had no apparent effect of cyclic electron flow. The inhibitor H Q N O binds to the plastoquinone-reducing Qc site o f C y t b 6 and blocks the so-called Q-cycle (Hauska et al. 1983). Like antimycin A, myxothiazol is a potent inhibitor of the Cyt bc complexes (Hauska et al. 1983) and acts in the chloroplast system as a specific inhibitor of chlororespiration (Ravenel and Peltier 1991). Similarly, no clear effect on ES in far-red light was observed upon inhibition of the ferredoxin-NADP+-reductase (FNR) by N-ethylmaleimide (NEM). N-Ethylmaleimide reacts with free sulphydryl groups on F N R which prevent the binding of ferredoxin (Shahak et al. 1981). However, N E M became a very effective inhibitor of PSI-cyclic ES when used in combination with antimycin A. The same was true for the cocktail N E M / H Q N O and NEM/myxothiazol. It was also observed that the combination of antimycin A and H Q N O (or myxothiazol) had no effect on ES in far-red light (data not shown) and that H Q N O , myxothiazol and antimycin A did not affect significantly the ES of linear electron flow in blue-green light. The observed synergism between N E M and antimycin/myxothiazol/ H Q N O demonstrates the existence of two alternative pathways for the in-vivo electron cycling around PSI: (i) an NEM-insensitive cycle (cycle I) in which electrons produced in PSI enter the plastoquinone pool via reduced ferredoxin through a route which is blocked by antimycin A, myxothiazol and H Q N O , and (ii) a myxothiazol/antimycin/HQNO-insensitive, NEM-sensitive cycle which involves the F N R (cycle II). This finding is in agreement with the previous in-vitro works on thylakoid fragments by Hossler and Yocum (1985, 1987) who showed the existence of an antimycin-insensitive cyclic photophosphorylation taking place when N A D P + was used as the final electron acceptor. Apparently, cycle I and cycle II are equally efficient in terms of maximal capacity of ES since the block of one cycle was fully compensated by the other cycle and vice versa. The light-saturation characteristics of the two cycles showed, however, significant differences. Whereas
Table 1. Effect of various chemicals on the maximal ES (extrapolated to the 0 light fluence rate as shown in Fig. 2) in far-red light (>715 nm, 17 Hz) and in broadband blue-green light (400-600 nm, 297 Hz) by Chlamydomonasreinhardtiicells. Data are expressed as fractions of the ES measured in untreated cells. On average, ES of control, untreated cells was 0.22 and 0.16 in far-red light and blue-green light, respectively. Data are means + SD of at least three experiments Treatments
ES (relative) in far-red in blue-green light light
No addition 10 ~tnaDCMU 20 ~tM DBMIB 40 ~tM PMA 1 mM pBQ 1 mM Methylviologen 40 laM Antimycin A 501aM Myxothiazol 10 I~M HQNO 800 taM NEM 800 pM NEM +40 taM antimycin A 150 laM NEM+50 laM myxothiazol 800 ~tM NEM+ 10 ~tM HQNO
1.00 0.98+0.05 0.0 0.05+_0.04 0.20_+0.04 0.34_+0.06 0.97 • 0.02 0.94_+0.11 1.05_+0.05 0.90+__0.14 0.11+0.03 0.14_+0.01 0.21+0.08
1.00 0.20_+0.04 0.0 0.16_+0.05 0.70• 0.51+0.13 1.06 • 0.11 0.89_+0.07 1.01 +0.18 0.60+0.11 0.31+0.07 0.35_+0.07 0.48+0.09
J. Ravenel et al. : The in-vivo cyclic electron pathways around PSI
255
the light-dependence curves of 1/ES in control cells and in antimycin-treated cells were similar, the N E M treatment markedly decreased the slope of the plot, indicating that cycle I (observed in the presence of NEM) was, less rapidly saturated with increasing irradiance than cycle II (monitored in the presence of antimycin; Fig. 3). The two electron cycles can also be distinguished on the basis of their PTo0 turnover time (Table 2). The turnover of Pv0o during cyclic electron transfer was quantified by determining the half-time (tl/2) of the dark re-reduction of P4oo (as monitored by cell absorbance changes at around 830 nm) after interrupting the far-red light illumination (Maxwell and Biggins 1976). In control, untreated Chlamydomonas cells, tlj2 was about 200 ms - a value close to that measured in various other algae by Maxwell and Biggins (1976). Inhibition of the cyclic electron flow by pBQ or NEM + antimycin A drastically rose the tl/2 value to about 3 s. Interestingly, tl/2 substantially decreased to approx. 75 ms in NEM-treated cells whereas antimycin A did not affect the postillumination recovery of Pv0o. From those kinetics data, one can conclude that (i) the turnover of P7oo was faster in cycle I than in cycle II, and (ii) the PToo turnover in the in-vivo cyclic pathway of untreated cells was close to that measured for cycle II, as were the light-saturation characteristics. Incidentally, it should be noticed that the rate of cyclic electron trans-
port is much slower (approx. 10 times slower) than that of non-cyclic electron transport (half-time of approx. 10-20 ms). The limiting step of the cyclic electron flow is therefore located in the return of reducing equivalents from PSI to plastoquinone. Although several previous reports (e.g. Forti and Zanetti 1969; Forti and Rosa 1971 ; Shahak et al. 1981) have reached the conclusion that there is a role for F N R in cyclic electron transport, the possible mode of action of the oxidoreductase is unclear. It has been suggested that F N R could play a regulatory role (Cleland and Bendall 1992), and could be an integral component of the cyclic pathway transferring electrons from reduced ferredoxin to plastoquinone (Hosler and Yocum 1985), to a plastoquinone reductase closely linked to cyt b 6 (Hind et al. 1981) or to NADP + (in cyanobacteria; Mi et al. 1992). In the following experiments, we have studied the effect of an NADP-analogue, 2'-monophosphoadenosine-5'diphosphoribose (PADR), on cyclic PSI activity of Chlamydomonas cells treated or not with antimycin A. 2'-Monophosphoadenosine-5'-diphosphoribose specific-
15
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Fig. 3. Effects of 400 gM N E M and 50 gM antimycin A (+ ANT) on the light-dependence curve of I/ES in Chlamydomonas cells. Energy storage in cells treated with both NEM and antimycin was dramatically reduced, resulting in 1/ES values out of the scale of this figure Table 2. Turnove of P7oo in PSI-driven cyclic electron transfer in Chlamydomonas reinhardtii cells illuminated with far-red light (730 nm), as estimated by the half-time (tl/2) of the dark reduction of Pv0o after switching off the far-red light. Data are means4. SD of three experiments Treatments
tl/2 (ms)
No additive 500 gM pBQ 1 mM NEM 50 gM antimycin A 1 mM NEM + 50 gM antimycin A
220444 41 3005 444519 76444 21 205 444 7 3153 + 140
+Fd + P A D R + A N T
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60
Ught fluence rate (W m2) Fig. 4. A Dependence of the reciprocal of ES on the (modulated) far-red light fluence rate in Chlamydomonas cells before (A) and after electroporation in the, absence (o) or presence (+ Fd, []) of ferredoxin (0.1 mg" ml-1). B Light-dependence curve of 1/ES in Chlamydomonas cells electroporated in the presence of ferredoxin (0.1 m g ' m 1 - 1 ) and 2.5mM PADR (+Fd+PADR, zx), 4 0 g M Antimycin A (+Fd+ANT, G) or 2.5 mM P A D R + 4 0 laM Antimycin A ( + Fd+ PADR + ANT, o)
256
J. Ravenel et al. : The in-vivo cyclic electron pathways around PSI
3. Maximal ES in far-red light (>715nm, 17Hz) in Chlamydomonas reinhardtii cells electroporated in the presence or absence of ferredoxin, PADR, NADP and antimycin A. Data are expressed as fractions of the ES measured in the cells supplemented with ferredoxin (1 = 29%). Data are means :t: SD of at least three independent experiments
Table
Treatments
ES (relative)
Treatments
ES
Before electroporation After electroporation without ferredoxin After electroporation with ferredoxin (0.1 mg" m1-1) After electroporation with ferredoxin (0.1 mg. m1-1) and 40 [aM antimycin A 2.5 mM PADR 2.5 mM PADR+ 40 [aM antimycin A 40 mM NADP
0.70 4-0.24 0.674- 0.23
Respiration
No additive Rotenone 50 [aM+ antimycin A 30 [aM Amobarbytal 200 laM+ HQNO 20 [aM Amobarbytat 400 raM+ antimycin A 40 [aM Acridone 200 [aM+antimycin A 40 [aM Hydroxypyridine 1 mM +antimycin A 40 [aM
1.00 0.93 1.00 1.00 1.10 1.00
1.00 0.73 0.79 0.60 0.66 0.68
Table
1.00
4. Effect of various inhibitors of mitochondrial NADHdehydrogenase on the maximal ES by the antimycin/HQNO-insensitive cyclic electron flow in Chlamydomonasreinhardtiicells illuminated with far-red light (>715 nm, 17 Hz). The effects of the inhibitors on mitochondrial respiration are also shown. Data are expressed as fractions of the values measured in the control cells
1.00:ko 1.00 • 0
0.59:k0.11 0.96
ally inhibits in a competitive manner the reduction of N A D P by F N R (Avron 1981). The uptake of this molecule by intact Chlamydomonas cells was induced by highvoltage electric pulses. Electropulsing is frequently used to introduce exogenous molecules such as D N A into various types of cells including intact Chlamydomonas reinhardtii cells (Brown et al. 1991). Figure 4A shows the dependence curve of 1/ES on the fluence rate of the exciting far-red light in control algal cells and in cells which were electroporated by two consecutive 20-ms pulses of 2 0 0 0 V . c m -1. Electroporation noticeably modified the slope of the plot (without concomitant change in the maximal ES value extrapolated to 0) showing faster saturation of PSI-mediated cyclic electron transfer with increasing fluence rate o f far-red light. This can be attributed to a loss of ferredoxin during electropulsing since addition of ferredoxin ( ~ 0.1 mg 9ml-1) to the electropermeabilization buffer cancelled the effect. Figure 4B showed the light-dependence curves of 1/ES in Chlamydomonas cells electroporated in the presence of ferredoxin and P A D R with or without antimycin A. Not unexpectedly, antimycin A or P A D R alone had no visible effect on ES. In contrast, the cocktail antimycin + P A D R significantly inhibited cyclic ES. These effects are quantified in Table 3. The addition of 40 gM antimycin A and 2.5 m M P A D R to the electroporation medium reduced the maximal ES by more than 40%. This finding indicates that, during (antimycin-insensitive) F N R dependent cyclic electron transport (cycle II), electrons are recycled into the plastoquinone pool via N A D P H . As the binding sites for ferredoxin and N A D P on F N R are believed to be independent (Zanetti 1976; Bookjans and B6ger 1979; Knaff et al. 1980), one can exclude the possibility that the binding of P A D R indirectly affects the F N R activity by perturbing the ferredoxin binding. Incidentally, the introduction of exogenous N A D P into the cells did not stimulate ES, suggesting that the N A D P concentration in vivo was not limiting for the cyclic electron flow around PSI. This does not seem to be the case for ferredoxin which had a positive effect on ES monitored in far-red light ( + 4 3 % on average) al-
though the extent of this stimulation was quite variable from one batch of cells to the other, as was the maximal ES value. Thus, the ferredoxin content of the cells might be a limiting factor for cyclic electron transport, depending on the physiological state and growth conditions of the algae. This problem is currently under investigation in our laboratory. The implication of N A D P H in cyclic electron transport through PSI raises the interesting possibility of an N A D P H dehydrogenase activity in the thylakoid membrane. In Table 4, we have examined the effects of various compounds known to inhibit the mitochondrial N A D H dehydrogenase (Oettmeier et al. 1992; Singer and Ramsay 1992): rotenone, amobarbytal, acridone and hydroxypyridine. Although all these inhibitors did penetrate into the cells and significantly reduced their rate of dark respiration as jugded by O2-exchange measurements with a Clark-type electrode, none of them were able to appreciably change the efficiency of ES by the antimycin-insensitive cyclic electron transfer. One can conclude from these data that the thylakoid NADPH-dehydrogenase, if present, is appreciably different from the mitochondrial N A D H dehydrogenase. Given the complexity of this latter enzymic system, containing not less than 26 subunits (Singer and Ramsay 1992), such a difference would not be very surprising.
Discussion
The scheme in Fig. 5 summarizes the main features of the in-vivo PSI-driven cyclic electron transport which have been established in the present work. At least two electron cycles (denoted I and II) operate in vivo. The part of the linear electron-transport chain located between plastoquinone and ferredoxin is common to both cycles, as shown by the complete suppression of ES in far-red light by the plastoquinol antagonist DBMIB and the ferredoxin competitors/inhibitors pBQ, methylviologen and PMA. Cycle I is virtually independent of the F N R activity and is likely to correspond to the ferredoxin-mediated cyclic electron flow previously observed in vitro which is sensitive to antimycin A (Tagawa et al. 1963). The exact site of action of this latter compound is unfortunately
J. Ravenel et al. : The in-vivo cyclic electron pathways around PSI Cycle
/ !
II
Cycle I :_:@ .......................................
NADPH
P/~R
~'~ "~Atb~
PQ (H~.___~.
cwtb~
J
c~
DBMIB
Fig. 5. Scheme of the pathways of cyclic electron transport in intact Chlamydomonas cells. Dashed arrows indicate electron-transfer reactions which are specific to the cyclic pathways. Wavy arrows indicate site of action of some inhibitors used in this study. NDH, NAD(P)H dehydrogenase; PQ(H)2, plastoquinol; Pc, plastocyanin; Fd, ferredoxin; FeS, the Rieske Fe-S protein; Anti and Myxo, antimycin A and myxothiazol. Other abbreviations are defined in the text
unknown. In contrast to mitochondrial and bacterial systems, neither antimycin A nor myxothiazol seem to bind to the quinone-reducing Qc site on the Cyt b6/f complex (as does HQNO; Moss and Bendall 1984; Ravenel and Peltier 1991). The inhibitory effect of HQNO demonstrates, however, that cycle I does require the operation of the protonmotive Q-cycle associated with Cyt b6. In fact, the mitchellian Q-cycle or its modified versions (see e.g. Bendall 1982; Crofts and Wraight 1983; Hauska et al. 1983; O'Keefe 1983) allow single electrons to enter the plastoquinone pool via Cyt b6. On the other hand, there are various reports showing that ferredoxin can reduce Cyt b6 in the dark (Arnon and Chain 1979; Telfer and Barber 1981; O'Keefe 1983) and, using isolated Cyt b6/f complexes, Lam and Malkin (1982) showed that this reduction is partially sensitive to antimycin A. The measured rates of Cyt b6 reduction by ferredoxin (O'Keefe 1983) are compatible with the cyclic electron-transport rates measured here, being approx. 10-30 times slower than the published rates of plastoquinone-plastocyanin oxidoreductase. Cycle II involves the (NEM-sensitive) F N R which catalyzes the (PADR-sensitive) reduction of NADP § by ferredoxin. Although redox interactions between stromal components and the photosynthetic intersystem carriers have been shown in chloroplasts of higher plants (Havaux et al. 1991 ; Asada et al. 1992), the direct implication of N A D P H in cyclic electron transport is shown here for the first time in eukaryotic cells. Actually, cycle II provides a simple NADPH-recycling mechanism, possibly involving a NAD(P)H-dehydrogenase, which allows the continuous adjustment of the NADPH/ATP ratio to the appropriate value needed in the stroma. The chloroplast genome has been shown to contain several genes (ndh) which are clearly homologous to genes encoding for mitochondrial N A D H dehydrogenase (complex I; Ohyama et al. 1988). These genes have been shown to be actively transcripted and some dehydrogenase subunits have been reported in chloroplasts of higher plants and
257
green algae (Matsubayashi et al. 1987; Peltier and Schmidt 1991; Berger et al. 1993). However, no clearly defined role in photosynthesis has been attributed so far to such an enzymatic complex. The fact that most of the ndh genes are missing from the plastid genome of the nonphotosynthetic parasitic plant Epifagus suggests, however, that its function is tied to photosynthetic metabolism (dePamphilis and Palmer 1990). It has been hypothesized that a NAD(P)H-plastoquinone oxidoreductase would be involved in the light-independent reduction of the plastoquinone pool associated with the (chloro)respiratory activity found in some photosynthetic eukaryotes including Chlamydomonas reinhardtii (Bennoun 1982; Verm6glio et al. 1990). Possibly, the chlororespiratory electron flow from NAD(P)H to Oz is a regulatory mechanism ensuring the recycling of NAD(P)H formed in the dark from starch breakdown by the glycolysis pathway (Bennoun 1982). From the data presented here, one can propose a function more closely related to photosynthesis as a means of maintaining PSI cyclic electron flow via NADPH. Such a role has already been suggested in cyanobacteria where the photosynthetic and respiratory electron-transport chains coexist in the same membrane and intersect at the site of the plastoquinone pool. In the cyanobacterium Synechocystis, lesions in the ndh genes were indeed observed simultaneously to cause the loss of the active transport of inorganic carbon (Ogawa 1991) - a process which is energized by the PSI-cyclic electron flow (Ogawa et al. 1985) - and a strong slow-down of the P7o0 turnover in far-red light (Mi et al. 1992). The chloroplastic N A D P H dehydrogenase appears, however, to be functionally different from the mitochondrial N A D H dehydrogenase, as judged by its insensitivity to various inhibitors of mitochondrial respiration. It is clear that further studies will have to identify and characterize this hypothetical chloroplastic dehydrogenase. This work is currently undertaken in our laboratory. Cycles I and II are two alternative pathways of cyclic electron transport with similar potential for photochemical energy storage but quite different turnover rate and light-saturation characteristics. Indeed, the light-dependence curve of ES and the PTo0 turnover time in far-red light were observed to be strongly affected by N E M (i.e. when only cycle I was functional) but not by antimycin A (when only cycle II was at work), thus suggesting that cycle II is the preferential in-vivo cyclic pathway under our experimental conditions. Hosler and Yocum (1987) have demonstrated that cyclic photophosphorylation of ADP by thylakoid membranes becomes insensitive to antimycin in the presence of NADP, presumably when the pools of ferredoxin and PT00 are largely oxidized. The latter condition is likely to prevail in Chlamydomonas cells exposed to moderate far-red light. Conversely, cyclic electron flow was observed to be antimycin-sensitive under reducing conditions (Hosler and Yocum 1987). Thus, one can see cycle I as a valve triggered when the capacity of PSI to recycle electrons via N A D P H is overflowed and electrons accumulate at the level of ferredoxin. An obvious advantage to divert electrons into this second route when the ferredoxin pool becomes reduced is the possi-
258
J. Ravenel et al. : The in-vivo cyclic electron pathways around PSI
bility o f reaching high rates o f cyclic p h o t o p h o s p h o r y l a tion u n d e r a wide range o f redox conditions. This flexibility m a y also be beneficial u n d e r strong light stress to avoid o v e r r e d u c t i o n o f the PSI acceptor side and subsequent p h o t o i n h i b i t i o n o f photosynthesis by superoxide p r o d u c e d f r o m reduced ferredoxin t h r o u g h the Mehler reaction (Mehler 1951) or f r o m reduced F N R and N A D P H t h r o u g h enzymatic processes ( M o r e h o u s e and M a s o n 1988). In this context, it also interesting to notice that antimycin A and m y x o t h i a z o l have been shown to affect b o t h cyclic PSI activity (this study) and chlororesp i r a t o r y O2 u p t a k e (Ravenel and Peltier 1991), suggesting that the two activities could be s o m e h o w related. Clearly, such a coupling o f the cyclic electron-transport system with the c h l o r o r e s p i r a t o r y oxidative p a t h w a y w o u l d constitute a useful m e a n s for recycling reducing equivalents o f photosynthesis. The rate and physiological significance o f chlororespiration remain, however, to be quantified in vivo. A n o t h e r i m p o r t a n t point which remains to be elucidated is the relative contribution o f cyclic PSI flow and other energizing/energy-dissipating pathways, such as the O z - d e p e n d e n t linear flow (Horm a n n et al. 1993), in light exciting b o t h photosystems.
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We are very grateful to Dr. M.-H. Montane (Cadarache, SaintPaul-lez-Durance, France) for her advice in the electroporation experiments.
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