cells were subjected to anaerobiosis by addition of glucose and glucose oxidase (25). Under these conditions, reduction of PQ pools is restricted to partial ...
Proc. Natl. Acad. Sci. USA Vol. 88, pp. 4791-4795, June 1991 Botany
Chlororespiration: An adaptation to nitrogen deficiency in Chlamydomonas reinhardtii (photosynthesis/chloroplast/thylakoid/NADH-plastoquinone oxidoreductase/cytochrome)
GILLES PELTIER*
AND
GREGORY W. SCHMIDT
Department of Botany, University of Georgia, Athens, GA 30602
Communicated by Martin Gibbs, February 14, 1991
When grown under nitrogen limitation, proABSTRACT nounced chlororespiratory activity develops together with an altered composition of thylakoid membranes in Chlamydomonas reinhardiii. Relative to control cultures, the flash-inhibited, chlororespiration-dependent 02 consumption signal increases 10-fold. Also augmented is the light-sensitive respiratory activity responsible for the "Kok effect," reflecting competitive inhibition of chlororespiratory electron transport by photosystem I. Fluorescence measurements show that the thylakoid plastoquinone pool is extensively reduced in dark-adapted, N-limited cells. Thylakoids of N-limited cells have reduced amounts of cytochrome b6, cytochromef, and light-harvesting complexes. However, thylakoid-bound NADH-PQ oxidoreductase, with major subunits of 51 kDa and 17 kDa, is increased 7-fold and two novel cytochromes of 34 and 12.5 kDa are highly abundant. Thus, components of photosynthetic and chlororespiratory electron transport pathways are differentially regulated by N availability.
Following the postulate of Goedheer (1) for the existence of chloroplast respiration, evidence for an oxidative electron transport pathway in thylakoids of green algae has steadily accumulated. Chlororespiration also might be present in vascular plants (2, 3) and chloroplasts contain genes encoding polypeptides similar to mitochondrial NADH oxidoreductases (4). Chlororespiratory constituents that presumably are involved in reduction of plastoquinone (PQ) pools, such as NADH-PQ oxidoreductase (5, 6) and succinate dehydrogenase (7), have been partially characterized in algal chloroplasts. Also, the redox states of PQ pools of green algae are affected by oxygen (8), selective respiratory inhibitors (8), and the products of starch metabolism (9). Recently, chlororespiratory oxygen uptake in green algae was demonstrated directly by using flash illumination and a combination of amperometric and mass spectrometric techniques (10). Chlororespiration is inhibited by myxothiazol and antimycin A, indicating the involvement of a cytochrome complex similar to cytochrome b/c complexes of bacteria or photosynthetic bacteria (36). Little else is known about the electron carriers or how PQ pools are oxidized. The physiological significance of a respiratory activity in photosynthetic membranes and whether chlororespiration is affected by environmental or developmental conditions have been unknown. In studies of the effects of N limitation on thylakoid biogenesis in Chlamydomonas, we have found that these conditions lead to a dramatic enhancement of chlororespiration. Moreover, N limitation induces changes of thylakoid composition: NADH-PQ oxidoreductase activity and the abundance of two novel cytochromes are markedly increased. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
MATERIALS AND METHODS Chlamydomonas reinhardtii 2137 was grown photoautotrophically in continuous culture systems that were continuously illuminated [150 AE m-2's-1; 1 E (einstein) = 1 mol of photons] and aerated without CO2 enrichment (11). Medium for N-limited (NL) cells contained 150 /LM NH4Cl as the sole nitrogen source whereas control cells were supplied with 7.5 mM NH4CI. Dilution rates were 0.15/day and 0.23/day for NL and control cultures, respectively. Flash-induced 02-exchange measurements employed a bare platinum electrode system similar to that of Schmid and Thibault (12). Cells harvested by low-speed centrifugation were resuspended in 50 mM Tris, pH 7.2/0.1 M KCI to provide conductivity for amperometric measurements. Within 15 min after addition to the electrode system, cells settle on the electrode to form an approximate monolayer. To maintain aerobic conditions and to compensate for rapid 02 consumption by the electrode and respiring cells, 02 was flushed at the gas/liquid interface of the sample. The chamber was covered by a conic reflector and a xenon flash (EG and G, FX 201, 2-.us duration) provided flash illumination. Net 02 exchange also was measured with a Clark-type 02 electrode (Rank Brothers, Cambridge, U.K.) with continuous illumination from a projector equipped with a heat filter (type KL 1500, Schott, Mainz, F.R.G). Chlorophyll fluorescence was measured using excitation light (430-450 nm) of a spectrofluorometer (SPF-SOOC, SLM Aminco, Urbana, IL) and a photodiode (type PIN 10 DP, United Detector Technology, Santa Monica, CA) protected with a red filter (89B, Kodak). Modulated fluorescence was measured using a pulse amplitude-modulated fluorometer (PAM-101, H. Walz, Effeltrich, F.R.G.) as described by Schreiber et al. (13). Illumination of samples and detection of fluorescence were accomplished via a fiber optic system. Thylakoids, purified as described (11), were resuspended in 60 mM Tris, pH 8.6/60 mM dithiothreitol/0.6 mM benzamidine/3 mM aminocaproic acid/12% sucrose and solubilized by addition of 2% lithium dodecyl sulfate. After heating for 30 sec at 950C, proteins were electrophoresed in 10-20% polyacrylamide gradient gels at 40C. Heme staining was according to Thomas et al. (14). For NADH-PQ dehydrogenase measurements, thylakoids were resuspended in 50 mM Tris'HCI, pH 7.5/0.5 mM EDTA and then were partially solubilized with 0.5% deoxycholate and 0.1% Triton X-100 for 30 min on ice. Dehydrogenase activity was measured by the NADH absorbance decrease at 340 nm in 50 mM Tris'HCI, pH 7.5/0.5 mM EDTA/0.1 mM NADH with 0.1 mM menadione as an electron acceptor. Nondenaturing PAGE was performed at 40C in a 5% polyacrylamide gel Abbreviations: NL, nitrogen-limited; LHC, light-harvesting complex; PQ, plastoquinone; PS, photosystem. *Permanent address: Departement de Physiologie Vegetale et tcosystemes, Centre d' Etudes de Cadarache, 13108 Saint-Paul-lezDurance, France.
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C"k-CAIMESPIRMURN
Proc. Natl. Acad. Sci. USA 88 (1991)
FUlt
PHOTOSYNTHETIC ELECTRON FLOW
FIG. 1. Diagram of photosynthetic and presumed chlororespiratory electron transport in thylakoids. PC, plastocyanin; FD, ferredoxin; FNR, ferredoxinNADP oxidoreductase.
containing 0.5% Triton X-100. The running buffer (pH 8.3) contained 50 mM Tris, 383 mM glycine, and 0.5% Triton X-100. Activity staining was carried out using p-nitroblue tetrazolium chloride (15). Protein determinations (16) employed bovine serum albumin as a standard. Chlorophyll was measured in 90%o methanol extracts (17).
RESULTS After growth in a NL continuous culture system, C. reinhardtii exhibits substantial changes in thylakoid protein profiles and photosynthetic carbon metabolism (refs.-12 and 18; D. B. Wilmot and G.W.S., unpublished data). To extend studies of adaptation to N availability, we evaluated the magnitude of chlororespiration in NL cells. The hypothetical flow of electrons in chlororespiratory and photosynthetic electron transport chains of thylakoids is shown in Fig. 1. Flash-induced 02 exchange was compared in NL and control cells by using a bare platinum 02 electrode (Fig. 2). After cells settled onto the electrode and adapted to darkness, an equilibrium state oflow 02 concentration was established: 02 consumption by the electrode and respiring cells was counterbalanced by 02 diffusion from the sample surface. Upon illumination with a series of short (2 us) and saturating flashes, damped oscillations of increasing 02 yields were observed (Fig. 2A). The large and rapid signals [ti/2(rise) = 10 ms] induced by the third and subsequent flashes resulted from water-splitting by photosystem (PS) 11 (19, 20). The smaller and slower 02 signals [tl/2(rise) = 350 ms] induced by the first two flashes resulted from inhibition of chlororespiration (10): PS I oxidizes PQ pools, thereby competitively inhibiting chlororespiratory electron transport to 02. With NL cells, chlororespiration-dependent signals relative to those of photosynthetic 02 evolution were about 10 times higher than in control cells (Fig. 2B). This indicates that
chlororespiration is augmented in response to NL growth conditions. Kok (21) noted a discontinuity in the yield of photosynthetic 02 evolution when cells were illuminated within a range oflow fluence. Subsequently, the "Kok effect" also has been documented as inhibition of chlororespiration because electron flow is diverted to PS I (22). Measurements of net 02 exchange as a function of light intensity shows that a substantial Kok effect occurs with NL cells but is very small in control algae (Fig. 3). At irradiance levels above 15 uE-m-2s-1, neither response slopes, reflecting photosynthetic quantum yields, nor maximum rates of photosynthesis are affected by N availability when measured relative to chlorophyll content (Fig. 3 Inset). In contrast, Fig. 3 and Table 1 show that, on a cellular dry weight basis, N limitation increases respiration rates 1.4- to 2.1-fold. Enhanced respiration accompanies a 7- to 8-fold diminution in chlorophyll concentration and photosynthetic activity per cell dry weight. As noted above, PQ pools are electron carriers for both photosynthetic and chlororespiratory electron transport (refs. 8, 10, and 23; Fig. 1). Therefore, enhanced chlororespiration should increase reduction of PQ pools, and this was verified by measurements of modulated fluorescence (Fig. 4). In these experiments, cells were illuminated by a weak modulated light source such that PQ was not significantly reduced by PS II. Consequently, minimal fluorescence, designated F0, was produced. When saturating actinic light of 500-ms duration was superimposed, fluorescence reached a maximum (Fmax) corresponding to 100%0 reduction of QA, the 2
0 ca 0
o" CM - 1 >0 0
A
B
o
i
L-
0
E -2 A A AA AA AA A AA A
A A A A A A A A A A A A
-3 2s
FIG. 2. Amperometric signals recorded following flash illumination (2 jzs) of control (A) and NL (B) Chlamydomonas cells deposited on a bare platinum 02 electrode. Algae were dark-adapted for 3 min before flashes were fired every 0.9 s.
0
10
20
30
40
Light Intensity (pE.m-2. sec-1 ) FIG. 3. Net 02 exchange measured as a function of light intensity
in control (-) and NL (o) cells. Cell suspensions were supplemented with 10 mM NaHCO3.
Proc. Natl. Acad. Sci. USA 88 (1991)
Botany: Peltier and Schmidt w U z w U Co
Table 1. Effect of N limitation on chlorophyll content, rates of respiration and photosynthesis, and thylakoid NADH-menadione
dehydrogenase activity in C. reinhardti Control 32.5 ± 5.6 Chlorophyll content* 3.17 ± 0.15 Respiration ratet 43.7 ± 4.3 Photosynthetic ratet NADH-menadione activityt 0.61 ± 0.14 per mg of dry weight. *lug tAumol of 02 per min per g of dry weight. *,umol of NADH per hr per mg of protein.
NL 4.24 ± 0.63 5.87 ± 0.75 7.97 ± 1.45 4.28 ± 0.80
N Limited -J U-
A
2OmV
(Fig. 4).
w 0 J
U-
LL
>c -j w
5mV 15s
The redox states of PQ pools regulate state transitions that result from reversible organization of light-harvesting complexes (LHCs) with PS II reaction centers (24, 25). To verify reduction of PQ pools by chlororespiration, we determined that N limitation also alters fluorescence states (Fig. 5). The maximum fluorescence of cells was measured immediately after they were removed from chemostats (initial state, Si, in Fig. 5). Subsequently, 3-(3,4-dichlorophenyl)-1,1-dimethylurea was added to block electron flow from PS II to PQ, and cells were subjected to anaerobiosis by addition of glucose and glucose oxidase (25). Under these conditions, reduction of PQ pools is restricted to partial reactions of chlororespiratory electron transport, inducing low-fluorescence state 2 (S2) as LHCs are dissociated from PS 11 (25). Illumination oxidizes PQ pools via PS I and fluorescence levels reach state 1 (S1) when LHCs reassociate with PS II. NL cells initially are in low-fluorescence states (S2) whereas control cells are mostly in high-fluorescence states (S1) (Fig. 5). This is consistent with the occurrence of more-reduced PQ pools in NL conditions. Note in Fig. 5 that the fluorescence changes associated with state transitions are about twice as fast in NL cells as in control cells. Enhanced chlororespiration in response to N limitation is accompanied by increased levels of presumed chlororespiratory electron carriers. NADH-PQ oxidoreductase of purified thylakoids solubilized with nonionic detergents was assayed by following NADH oxidation in the presence of menadione, a PQ analog. NADH-menadione oxidoreductase activity was -7-fold higher (on a protein basis) in NL cells than in control cells (Table 1). Solubilized membranes also were centrifuged 60 min at 100,000 x g and supernatants were subjected to nondenaturing PAGE in the presence of 0.5% Triton X-100. Upon staining for NADH dehydrogenase activity, a single band of intense activity was visualized with preparations from NL cells (Fig. 6). Studies with electron transport inhibitors have indicated that unique cytochromes might be involved in chlororespi12 w 251 F z LLJ
Control
0
primary PS II electron acceptor. The FO/Fn= ratio, a measure of the extent to which QA remained reduced because secondary electron acceptors (PQ pools) were unavailable, was about 1.5 times higher in NL cells than in control cells
C.)
4793
,.I-SS A Wsi ~ AS -s 15 t,,2-1 02s t112-1 80s
FIG. 4. Chlorophyll fluorescence levels in control and NL cells. Modulated fluorescence was measured directly with cells in chemostats using a fiber optic system. Chemostats were temporarily placed in the dark. Subsequently, a weak modulated light was employed to determine F0 and saturating flashes (500-ms duration) were superimposed to determine Fc. Note differences in fluorescence intensity scales.
ration in Chlamydomonas (36). To determine whether thylakoid cytochromes are modified in response to N availability, we analyzed heme-associated peroxidase activities of thylakoid proteins separated by SDS/PAGE. Four stained bands were detected from both NL and control membranes: cytochromef, cytochrome b6 and two unidentified bands, h, and h2, previously observed by Lemaire et al. (26). Whereas cytochrome f and cytochrome b6 were more abundant in control cells than in NL cells, h1 and h2 were especially abundant in thylakoids from NL cells. The protein-compositions of thylakoids of control and NL cells were compared by SDS/PAGE (Fig. 7). Apoproteins of LHCs (21, 23, 26, and 28' kDa) were diminished in NL thylakoids (see also ref. 11). However, bands that were more intense in NL thylakoids included two corresponding in size to cytochromes h1 and h2 (34 and 12.5 kDa) as well as at least eight unidentified polypeptides (arrowheads). An excised band of NADH-PQ oxidoreductase activity (as shown in Fig. 6) was subjected to second-dimension SDS/PAGE (Fig. 7). A major component of 51 kDa and a minor one of 17 kDa were identified, corresponding in size to two polypeptides of unfractionated thylakoids whose abundance was increased by NL growth conditions (Fig. 7). These polypeptides were similar in size to those (52 kDa and 17 kDa) reported by Aipes et al. (27) for NADH dehydrogenase of the cyanobacterium Anabaena variabilis.
DISCUSSION Depending upon nutrient availability, green algal cells adjust the balance between photosynthetic and respiratory activities
20
B
Si
t,,2-36s
10-
5 0
a0
11 ,, 10
20
30
TIME (min)
40
50
0
10
20
TIME
30
((min)
40
FIG. 5. Fluorescence changes associated with state transitions in control (A) and NL (B) cells. After removal from chemostats, cells were placed in the dark in an 02 electrode cuvette. Fm. was measured every 20 s, using saturating actinic flashes (500-ms duration). Initial Fn (Si) was determined and, at times indicated by breaks in the graph, 3-(3,4dichlorophenyl)-1,1-dimethylurea (10 ,M) and glucose/glucose oxidase were 5o added to induce transition to state 2 (S2). Transition to state 1 (Si) was induced by continuous illumination (open bar at top).
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Proc. Natl. Acad. Sci. USA 88 (1991)
A
NL
B
C
C
NL
cyt. f
h cyt.
b6
FIG. 6. (A) Heme staining of polypeptides from purified thylakoids after SDS/PAGE. (B) NADH dehydrogenase activity staining of thylakoids complexes after nondenaturing electrophoresis. In both experiments, equal amounts of protein from control (C) or NL thylakoids were used. cyt., Cytochrome.
of thylakoids. In N-limiting conditions, thylakoids contain decreased amounts of photosynthetic components such as LHC I and LHC 11 (11, 18) and the cytochrome b6/fcomplex (Fig. 6). Conversely, chlororespiratory components are present in higher concentrations. N deficiency has been reported to cause substantial changes in both chlorophyll content and photosynthetic activity (11, 28, 29). In addition, N deficiency is generally accompanied by accumulation of large amounts of starch and lipids (refs. 18, 29, and 30; D. B. Wilmot and G.W.S., unpublished data). High levels of potential respiratory substrates correlate with an =;2-fold stimulation of total respiration (Table 1). Based upon studies with inhibitors of mitochondrial electron transport (22), mitochondrial respiration is light-insensitive. Thus, chlororespiration can be estimated from the extent that total cell respiration is inhibited by low-intensity illumination (i.e., the Kok effect) (22). Because control cells exhibit a relatively insignificant Kok effect (Fig. 3), their respiration is almost exclusively mitochondrial. In contrast, the contribution of chlororespiration in NL cells is at least 30%o of total respiration (-1.8 Amol of 02 per min per g of dry weight). Mitochondrial respiration would be responsible for the remaining part of respiration in NL cells and it is about 1.3-fold higher than in control cells. Therefore, the overall effect of N limitation on respiratory activities is a large
ox
NL C
z1: 43-
29
hi,34 -
28 26
.......
l18. 4 14.3
--23
:-
21
-17C ..17_
-
h2.12.5-
FIG. 7. (Left) Polypeptide patterns of thylakoids from control (C) and NL Chlamydomonas. Arrowheads indicate bands in SDS/ polyacrylamide gels whose staining intensity was significantly modified in response to N availability. (Right) Second-dimension SDS/ polyacrylamide gel of the tIADH oxidoreductase (OX) activity band excised from the gel of Fig. 6B. Dashes indicate molecular size standards of 43, 29, 18.5, and 14.3 kDa.
increase of chlororespiratory activity coupled with a modest stimulation of mitorespiration. Increased chlororespiration in NL cells is not simply due to a higher electron flow rate through a constitutive pathway. Instead, NL thylakoids contain greater amounts of chlororespiratory electron carriers, as exemplified by higher NADH-PQ oxidoreductase activities ofthese membranes. In addition, two cytochromes, h1 and h2, suggested by Lemaire et al. (26) to be involved in chlororespiration are more abundant in NL cells. Inhibitory effects of myxothiazol and antimycin A on chlororespiration indicate the occurrence of cytochromes specific to chlororespiration thylakoids of Chlamydomonas (36). Our results further implicate h1 and h2 as components of the chlororespiratory chain. Especially upon developing high rates of chlororespiration, electron transport activities of Chlamydomonas thylakoids resemble those of cyanobacteria and photosynthetic bacteria. In these organisms, photosynthetic and respiratory activities can occur in the same membrane and their interactions account for inhibition of 02 uptake by light (31). Moreover, depending on conditions of growth (such as 02 availability or light intensity), photosynthetic bacteria differentially synthesize respiratory or photosynthetic electron transfer components (32, 33). The parallels of chlororespiration in NL Chlamydomonas may extend to characteristics of the electron transport components: the sizes ofthe major subunits ofthe NADH dehydrogenase in algal thylakoids are similar to those of cyanobacteria (Fig. 7 and ref. 27). Under the assumption that chloroplasts arose from endosymbiotic events between an ancestral cyanobacterium and a eukaryote, chlororespiration as well as photosynthesis could be of prokaryotic origin. Whether the chlororespiratory chain is also present in chloroplasts of higher plants is an important question. Sequencing of chloroplast genomes of tobacco and liverwort has revealed coding regions with high homology to genes coding for mitochondrial NADH dehydrogenase subunits (4). The corresponding gene products are candidates for participation in chlororespiration during conditions of N deficiency. Although the oxidizing components of chlororespiration are abundant' in cells grown in N-limiting conditions, their levels do not appear to be sufficient to completely oxidize pools of reduced PQ. In dark-adapted cells, NADH-PQ oxidoreductase appears to be more highly active, so that PQ pools are in a sustained state of reduction. When PQ pools are reduced, a kinase responsible for phosphorylation of LHC II is activated (24, 34), inducing migration of LHCs initially associated with PS II (state 1) towards PS I. This results in the low-fluorescence state (state 2). NL Chiamydomonas cells are in state 2 (Fig. 4) due to steady-state reduction of PQ
pools by chlororespiration. An interesting feature of N limitation is that the efficiency of the light reactions of photosynthesis are not strongly affected when measured on a chlorophyll basis (Fig. 3). Neither quantum yields, estimated from the slope of irradiance response curves, nor maximum rates of photosynthesis are influenced by N availability. This is surprising because the pigment organization of thylakoids from NL cells is substantially modified; LHC II levels are reduced and LHC I is almost completely missing from NL thylakoids (refs. 11 and 18; unpublished data). Unusual photosynthetic characteristics have also been reported for spinach plants supplied with different concentrations of nitrate (28). Our 'studies indicate mobile LHCs associate primarily with PS I in thylakoids of NL cells, causing state 2. This may partially compensate for the deficiency of LHC I antenna. Some ofthe differences in the kinetics of fluorescence changes related to state transitions (Fig. 5) can be explained by the lower LHC II content of NL cells (see ref. 11): if levels of the kinase and phosphatase that drive state transitions do not change but
Botany: Peltier and Schmidt substrate LHC II concentrations are decreased, state transitions should occur more rapidly. Why do algal cells respond to a deficiency in nitrogen by increasing chlororespiration? In adapting to such conditions, photosynthetic carbon metabolism becomes directed toward accumulation of extravagant amounts of starch and lipids (29, 30). We do not know whether chlororespiration arises subsequent to carbohydrate accretion or if it is more directly induced by N limitation. Nonetheless, chlororespiration, by facilitating carbohydrate oxidation (9) and/or NAD(P)H oxidation, might be a device to recycle the reducing equivalents of photosynthesis. One apparent benefit would be the contribution of chlororespiration to ATP synthesis. Perhaps more importantly, we suppose NADPH in NL cells cannot be efficiently oxidized via the synthesis of amino acids, chlorophyll, and nucleic acids. Furthermore, NADPH-driven CO2 reduction appears to reach saturation upon formation of massive amounts of storage carbohydrates in NL cells. Chlororespiration could be essential for maintaining NADP+ availability for photosynthetic electron transport. Thereby, it would attenuate photoinhibition and/or prevent oxygen toxicity due to nonenzymatic generation of superoxide by reduced ferredoxin (Mehler reaction) and enzymatic synthesis of superoxide and hydroxyl radicals by reduced ferredoxinferredoxin:NADP+ oxidoreductase and NADPH (35).
Proc. Natl. Acad. Sci. USA 88 (1991)
4795
We thank Dr. R. Sage for the use of the modulated fluorometer. This work was supported by Award DE-FG09-84ER13188 from the Department of Energy. G.P. was supported by the Commissariat a l'Energie Atomique, France.
10. Peltier, G., Ravenel, J. & Vermeglio, A. (1987) Biochim. Biophys. Acta 893, 83-90. 11. Plumley, F. G. & Schmidt, G. W. (1989) Proc. Nati. Acad. Sci. USA 86, 2678-2682. 12. Schmid, G. H. & Thibault, P. (1979) Z. Naturforsch. 34, 414-418. 13. Schreiber, U., Schliva, U. & Bilger, W. (1986) Photosyn. Res. 10, 51-62. 14. Thomas, P. E., Ryan, D. & Levin, W. (1976) Anal. Biochem. 75, 168-176. 15. Scherer, S., Alpes, I., Sadowski, H. & Boger, P. (1988) Arch. Biochem. Biophys. 267, 228-235. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 17. Peltier, G. & Thibault, P. (1985) Plant Physiol. 79, 225-230. 18. Plumley, F. G., Douglas, S. E., Switzer, A. B. & Schmidt, G. W. (1989) in Photosynthesis, ed. Briggs, W. R. (Liss, New York), pp. 311-329. 19. Joliot, P., Barbieri, G. & Chabaud, R. (1969) Photochem. Photobiol. 10, 309-329. 20. Kok, B., Forbush, B. & McGloin, M. (1970) Photochem. Photobiol. 11, 457-475. 21. Kok, B. (1949) Biochim. Biophys. Acta 3, 625-631. 22. Peltier, G. & Sarrey, F. (1988) FEBS Lett. 228, 259-262. 23. Bennoun, P. (1983) FEBS Lett. 156, 363-365. 24. Allen, J. F., Bennett, J., Steinback, K. E. & Amtzen, C. J. (1981) Nature (London) 291, 21-25. 25. Delepelaire, P. & Wollman, F. A. (1985) Biochim. Biophys. Acta 809, 277-283. 26. Lemaire, C., Girard-Bascou, J., Wollman, W. A. & Bennoun, P. (1986) Biochim. Biophys. Acta 851, 229-238. 27. Alpes, I., Scherer, S. & Boger, P. (1989) Biochim. Biophys.
1. Goedheer, J. C. (1963) Biochim. Biophys. Acta 66, 61-71. 2. Garab, G., Lajko, F., Mustardy, L. & Marton, L. (1989) Planta 179, 349-358. 3. Vermeglio, A., Ravenel, A. & Peltier, G. (1990) in Recent Advances in Experimental Phycology, eds. Wiessner, W., Robinson, D. G. & Starr, R. C. (Springer, Berlin), Vol. 7, pp. 188-205. 4. Ohyama, K., Kohchi, T., Sano, T. & Yamada, Y. (1988) Trends Biochem. Sci. 13, 19-22. 5. Godde, D. & Trebst, A. (1980) Arch. Microbiol. 127, 245-252. 6. Godde, D. (1982) Arch. Microbiol. 131, 197-202. 7. Willeford, K. O., Gombos, Z. & Gibbs, M. (1989) Plant Physiol. 90, 1084-1087. 8. Bennoun, P. (1982) Proc. Nati. Acad. Sci. USA 79, 4352-4356. 9. Gfeller, R. P. & Gibbs, M. (1985) Plant Physiol. 77, 509-511.
Acta 973, 41-46. 28. Evans, J. R. & Terashima, I. (1987) Aust. J. Plant Physiol. 14, 59-68. 29. Saux, C., Lemoine, Y., Marion-Poll, A., Valadier, M. H., Deng, M. & Morot-Gaudry, J. F. (1987) Plant Physiol. 84, 67-72. 30. Gallagher, R. N. & Brown, R. H. (1977) Crop Sci. 17, 85-88. 31. Scherer, S., Almon, H. & Boger, P. (1988) Photosyn. Res. 15, 95-114. 32. Lampe, H. H. & Drews, G. (1972) Arch. Mikrobiol. 84, 1-19. 33. La Monica, R. F. & Marrs, B. L. (1976) Biochim. Biophys. Acta 423, 431-439. 34. Wollman, F.-A. & Delepelaire, P. (1984) J. Cell. Biol. 98, 1-7. 35. Morehouse, K. M. & Mason, R. P. (1988) J. Biol. Chem. 263, 1204-1211. 36. Ravenel, J. & Peltier, G. (1991) Photosynth. Res., in press.
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