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Photosystem I Is Indispensable for Photoautotrophic Growth, CO2 Fixation, and H2 Photoproduction inChlamydomonas reinhardtii

1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês

10.1074/jbc.274.15.10466

1083-351X

Kevin Redding, Laurent Cournac, Ilya R. Vassiliev, John H. Golbeck, Gilles Peltier, Jean-David Rochaix,

Algal biology and biofuel production

Certain Chlamydomonas reinhardtiimutants deficient in photosystem I due to defects in psaAmRNA maturation have been reported to be capable of CO2fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L., and Mets, L. J. (1995) Nature 376, 438–441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364–367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of eitherpsaA or psaC; these activities could be restored to the F8-psaAΔ mutant by reintroduction ofpsaA. We observed limited O2photoevolution in mutants lacking photosystem I; use of18O2 indicated that this O2evolution is coupled to O2 uptake (i.e.respiration) rather than CO2 fixation or H2evolution. We conclude that the reported instances of CO2fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I. Certain Chlamydomonas reinhardtiimutants deficient in photosystem I due to defects in psaAmRNA maturation have been reported to be capable of CO2fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L., and Mets, L. J. (1995) Nature 376, 438–441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364–367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of eitherpsaA or psaC; these activities could be restored to the F8-psaAΔ mutant by reintroduction ofpsaA. We observed limited O2photoevolution in mutants lacking photosystem I; use of18O2 indicated that this O2evolution is coupled to O2 uptake (i.e.respiration) rather than CO2 fixation or H2evolution. We conclude that the reported instances of CO2fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I. photosystem I and II, respectively primary donor of PS I Tris acetate/phosphate medium high salt minimal agar medium wild type (or wild-type) The proposal that two different light reactions are involved in oxygenic photosynthesis arose from studies of an "enhancement effect" that was observed when two beams of different wavelengths were used to illuminate algae (3Emerson R. Annu. Rev. Plant Physiol. 1958; 9: 1-24Crossref Google Scholar, 4Myers J. Annu. Rev. Plant Physiol. 1971; 22: 289-312Crossref Google Scholar). The discovery of cytochromesb and f led to the concept that they served as intermediaries between the two photosystems, which operated in series to achieve linear electron transfer from H2O to NADP+ (5Hill R. Bendall F. Nature. 1960; 186: 136-137Crossref Scopus (344) Google Scholar). The essence of this "Z-scheme" model is that photosystem II (PS II)1accomplishes the oxidation of water and the reduction of plastoquinone and cytochromes but is unable to reduce ferredoxin or NADP+; photosystem I (PS I) is required for the reduction of NADP+ and the oxidation of cytochrome fthrough the mediation of the soluble protein plastocyanin. Although other explanations have been proffered for the enhancement effect and photosystem cooperativity (6Arnon D.I. Photosynth. Res. 1995; 46: 47-71Crossref PubMed Scopus (21) Google Scholar), the Z-scheme is strongly supported by experimental data (7Duysens L.N.M. Amesz J. Kamp B.M. Nature. 1961; 190: 510-511Crossref PubMed Scopus (155) Google Scholar) and is currently considered as the "central dogma" of oxygenic photosynthesis (8Trebst A. Annu. Rev. Plant Physiol. 1974; 25: 423-458Crossref Google Scholar, 9Stryer L. Biochemistry. 4th Ed. W. H. Freeman and Co., New York1995: 653-682Google Scholar). However, photosynthetic CO2 fixation and photoautotrophic growth were recently reported (1Greenbaum E. Lee J.W. Tevault C.V. Blankinship S.L. Mets L.J. Nature. 1995; 376: 438-441Crossref Scopus (69) Google Scholar, 2Lee J.W. Tevault C.V. Owens T.G. Greenbaum E. Science. 1996; 273: 364-367Crossref PubMed Scopus (24) Google Scholar) in two PS I-deficient mutants, B4 and F8, both of which are nuclear mutants deficient in thetrans-splicing of the psaA mRNA (1Greenbaum E. Lee J.W. Tevault C.V. Blankinship S.L. Mets L.J. Nature. 1995; 376: 438-441Crossref Scopus (69) Google Scholar, 10Girard J. Chua N.H. Bennoun P. Schmidt G. Delosme M. Curr. Genet. 1980; 2: 215-221Crossref PubMed Scopus (65) Google Scholar, 11Goldschmidt-Clermont M. Girard-Bascou J. Choquet Y. Rochaix J.D. Mol. Gen. Genet. 1990; 223: 417-425Crossref PubMed Scopus (115) Google Scholar). Using a gas flow apparatus, Greenbaum et al. (1Greenbaum E. Lee J.W. Tevault C.V. Blankinship S.L. Mets L.J. Nature. 1995; 376: 438-441Crossref Scopus (69) Google Scholar) were able to measure an evolution of O2 coupled to CO2fixation or to H2 evolution. The amount of CO2fixed by these mutant strains was roughly equivalent to that measured in wild-type (WT) cells. Although CO2 fixation in the PS I-deficient strains initially appeared to be limited to anaerobic conditions (1Greenbaum E. Lee J.W. Tevault C.V. Blankinship S.L. Mets L.J. Nature. 1995; 376: 438-441Crossref Scopus (69) Google Scholar), photoautotrophic growth was later reported in the presence of O2 (2Lee J.W. Tevault C.V. Owens T.G. Greenbaum E. Science. 1996; 273: 364-367Crossref PubMed Scopus (24) Google Scholar). Several control experiments were carried out by the authors to eliminate the possibility of trace amounts of PS I. Irradiation with far red light (λ > 700 nm), which excites PS I but not PS II, produced a small amount of H2evolution in WT cells but not in the B4 mutant (1Greenbaum E. Lee J.W. Tevault C.V. Blankinship S.L. Mets L.J. Nature. 1995; 376: 438-441Crossref Scopus (69) Google Scholar). Photobleaching experiments also failed to detect P700 in thylakoid membranes from these mutants (2Lee J.W. Tevault C.V. Owens T.G. Greenbaum E. Science. 1996; 273: 364-367Crossref PubMed Scopus (24) Google Scholar). The core of PS I is made up of the two largest subunits, PsaA and PsaB. They bind all of the cofactors involved in intra-PS I electron transport with the exception of the terminal iron-sulfur clusters, which are bound by the extrinsic PsaC subunit. Methods to delete the chloroplast genes psaA, psaB, andpsaC, have been described recently (12Fischer N. Stampacchia O. Redding K. Rochaix J.D. Mol. Gen. Genet. 1996; 251: 373-380Crossref PubMed Scopus (142) Google Scholar). Such mutants were incapable of CO2 fixation or photoautotrophic growth, but reintroduction of the deleted gene restored photoautotrophic growth (13Cournac L. Redding K. Bennoun P. Peltier G. FEBS Lett. 1997; 416: 65-68Crossref PubMed Scopus (19) Google Scholar, 14Fischer N. Setif P. Rochaix J.D. Biochemistry. 1997; 36: 93-102Crossref PubMed Scopus (73) Google Scholar, 15Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.-D. EMBO J. 1998; 17: 1-11Crossref PubMed Scopus (85) Google Scholar). Two hypotheses can be formulated to explain the discrepancy between the results with deletion mutants and trans-splicing mutants. On the one hand, the B4 and F8 strains could have harbored undetected amounts of PS I that allowed synthesis of enough NADPH to fix CO2 and grow photoautotrophically. On the other hand, the B4 and F8 strains could have possessed an as yet unidentified system that allowed PS I-independent reduction of NADP+. We have undertaken an analysis of several different PS I-deficient mutants (see Fig. 1) to examine critically the claim that PS I-deficient mutants can perform photosynthesis. We have used immunoblots to detect PS I subunits as well as spectroscopic means to measure photooxidizable P700, the primary electron donor of PS I (for reviews on PS I, see Refs. 16Golbeck J.H. Bryant D.A. Lee C.P. Current Topics in Bioenergetics: Light Driven Reactions in Bioenergetics. 16. Academic Press, Inc., New York1991: 83-177Google Scholar and 17Brettel K. Biochim. Biophys. Acta. 1997; 1318: 322-373Crossref Scopus (438) Google Scholar). We have made use of real time mass spectroscopy to measure the rates of O2 photoevolution, CO2 fixation, and H2 photoevolution. Our results indicate very clearly that photosynthesis requires the presence of active PS I. Our WT strain was isolated as an mt+ segregant from a cross between two derivatives of the 137c strain (18Harris E.H. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, Inc., San Diego1989Google Scholar). The original deletions of psaA andpsaC were made in this strain (12Fischer N. Stampacchia O. Redding K. Rochaix J.D. Mol. Gen. Genet. 1996; 251: 373-380Crossref PubMed Scopus (142) Google Scholar). Isolates of the F8 strain (10Girard J. Chua N.H. Bennoun P. Schmidt G. Delosme M. Curr. Genet. 1980; 2: 215-221Crossref PubMed Scopus (65) Google Scholar) were kindly provided by Dr. E. Greenbaum (Oak Ridge National Laboratory) and from the Chlamydomonas Stock Center (Duke University). The FUD26 strain (19Girard-Bascou J. Choquet Y. Schneider M. Delosme M. Dron M. Curr. Genet. 1987; 12: 489-495Crossref PubMed Scopus (56) Google Scholar, 20Girard-Bascou J. Curr. Genet. 1987; 12: 483-488Crossref Scopus (25) Google Scholar) was obtained from Dr. J. Girard Bascou (Institut de Biologie Physico-Chimique) and Dr. L. Mets (University of Chicago). The B4 strain (1Greenbaum E. Lee J.W. Tevault C.V. Blankinship S.L. Mets L.J. Nature. 1995; 376: 438-441Crossref Scopus (69) Google Scholar) was kindly provided by Dr. L. Mets. Bioballistic chloroplast transformations were performed with plasmids designed to delete the psaA and psaC genes, and homoplasmicity of these deletions was assessed by polymerase chain reactions (12Fischer N. Stampacchia O. Redding K. Rochaix J.D. Mol. Gen. Genet. 1996; 251: 373-380Crossref PubMed Scopus (142) Google Scholar). The aadA cassettes used to deletepsaA or psaC were flanked by direct repeats; homologous recombination between them removes the aadA cassette, leaving one repeat behind (12Fischer N. Stampacchia O. Redding K. Rochaix J.D. Mol. Gen. Genet. 1996; 251: 373-380Crossref PubMed Scopus (142) Google Scholar). After several subcloning steps on medium without antibiotic, spectinomycin-sensitive isolates were obtained; loss of aadA was confirmed by specific polymerase chain reactions (12Fischer N. Stampacchia O. Redding K. Rochaix J.D. Mol. Gen. Genet. 1996; 251: 373-380Crossref PubMed Scopus (142) Google Scholar). All experiments herein used theaadA-less strains to eliminate any differences due to chloroplastic expression of the aminoglycoside adenyltransferase enzyme. The third exon of psaA was reintroduced by bombarding psaAΔ mutants with plasmid pKR137 (15Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.-D. EMBO J. 1998; 17: 1-11Crossref PubMed Scopus (85) Google Scholar), which carries psaA exon 3 with the psbD-aadA-rbcLcassette (21Nickelsen J. van Dillewijn J. Rahire M. Rochaix J.D. EMBO J. 1994; 13: 3182-3191Crossref PubMed Scopus (126) Google Scholar) inserted at the EcoRI site approximately 300 base pairs downstream of psaA exon 3. During characterization of one of the psaAΔ deletion mutants of B4 (B4-psaAΔ-2), we isolated a photoautotrophic revertant able to grow on minimal medium. This clone, renamed B4-r2, was subsequently found to express PsaA and was not homoplasmic forpsaAΔ. Further subcloning of B4-psaAΔ-2 on antibiotic medium produced clones homoplasmic for psaAΔ. We attempted to isolate photoautotrophic revertants of B4-psaAΔ-2 in three separate trials by spreading cells on high salt minimal agar medium (HSM; 9 × 105 cells cm−2) and illuminating them (30–60 μmol of photons m−2 s−1) either aerobically or anaerobically. For some of these experiments, we UV-irradiated the HSM plates (8 mJ cm−2 of 260-nm radiation) immediately after plating the cells and held them in the dark for 24–48 h before illumination in an attempt to increase the reversion frequency. In total, 3.7 × 109 cells were tested (1.7 × 109 cells without UV and 2.0 × 109 cells with UV treatment), and no photoautotrophic revertants were isolated. However, B4 appears to revert at a low rate, and we have succeeded in isolating only two photoautotrophic revertants from it. Tris acetate/phosphate medium (TAP) and HSM for heterotrophic and photoautotrophic growth, respectively, were prepared as described (18Harris E.H. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, Inc., San Diego1989Google Scholar). Chlamydomonas reinhardtii cultures were maintained at 25 °C. In general, 100-ml TAP cultures were shaken at 180 rpm in 500-ml Erlenmeyer flasks under low illumination (∼1 μmol of photons m−2 s−1). For growth tests, 12 μl of log phase cultures were spotted onto agar media. TAP plates were kept under low light ( 760 nm) and was measured at the time constant of 3 ms. The actinic light from a 300-watt tungsten lamp was filtered using a hot mirror and a red cut-off filter to provide a broad band peaking at 715 nm with a full-width half-maximum of 40 nm and an intensity of 60 μmol of photons m−2 s−1, incident on the sample at a right angle to the probe beam. The actinic light was controlled with a shutter (Vincent Associates, Rochester, NY; 2-ms full opening/closing time). The absorbance changes (ΔA) were analyzed using IgorPro (WaveMetrics, Lake Oswego, OR), and their amplitudes were calculated as 0.434*ΔI/I, where I is the intensity of probe beam, and ΔI is the actinic light-induced change. Due to variations in sample densities (in the range of 1.6–7 × 107 cells ml−1), the ΔA signals were normalized to cell density. Algal cultures were harvested, and 1.5 ml of the suspension was placed in the measuring chamber. Dissolved gases were directly introduced in the ion source of the mass spectrometer (model MM 14–80, VG instruments, Cheshire, United Kingdom) through a Teflon membrane. For O2exchange measurements, the sample was sparged with N2 to remove 16O2, and 18O2(95% 18O isotope content; Euriso-Top; Saint Aubin, France) was then introduced to reach an O2 concentration close to the equilibrium with air. CO2 exchange measurements were performed following the NaH13CO3 addition (0.3 mm final concentration; 99% 13C isotope content; Euriso-Top) in the dark before recording CO2exchange. Light was supplied by a fiber optic illuminator (Schott, Main, Germany), and neutral filters were used to vary light intensity. Unless specified, experiments shown here were performed at 300 μmol of photons m−2 s−1 incident light. All gas exchange measurements were performed at 25 °C. The hydrogenase enzyme is known to be inactivated by oxygen and to require anaerobic conditions for its induction. Thus, for measurements of H2 evolution, cultures were first subjected to anaerobiosis by sparging the sample with N2, closing the chamber, and letting the algae consume O2 from the medium. The O2 concentration was monitored by mass spectrometry until it became undetectable (<0.2 μm), and the cultures were incubated for a further 30 min. In companion experiments, glucose (20 mm final concentration) and glucose oxidase (2 mg/ml final concentration) were added to verify that O2 depletion before illumination was complete and that hydrogenase induction and activity were not affected by residual oxygen. To introduce the methods used in this work, we will first describe characterization of the WT strain (see "Experimental Procedures"), which can grow photoautotrophically as well as heterotrophically on acetate-containing medium in the dark (Fig.2 A). Using specific antibodies against either PsaA (an integral membrane core subunit) or PsaD (a stromal subunit involved in docking ferredoxin to the acceptor side of PS I; Refs. 25Hanley J. Setif P. Bottin H. Lagoutte B. Biochemistry. 1996; 35: 8563-8571Crossref PubMed Scopus (42) Google Scholar and 26Lelong C. Setif P. Lagoutte B. Bottin H. J. Biol. Chem. 1994; 269: 10034-10039Abstract Full Text PDF PubMed Google Scholar), we could easily visualize these polypeptides in membranes prepared from the WT strain (Fig. 2 A). The in vivo fluorescence induction kinetics (Fig. 2 B) are typical of normal algae (27Bennoun P. Chua N.H. Bücher T. Neupert W. Sebald W. Werner S. Genetics and Biogenesis of Chloroplasts and Mitochondria. North-Holland Publishing Co., Amsterdam1976: 33-39Google Scholar). The rise in fluorescence, which is correlated with reduced QA in PS II, is due to the reduction of the plastoquinone pool, while the subsequent drop has been interpreted as originating from oxidation of the plastoquinone pool due to the combined action of PS I and cytochrome b 6 f (28Krause G. Weis E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 313-349Crossref Scopus (3695) Google Scholar). In order to measure PS I quantitatively, we monitored the kinetics and extent of photooxidation and dark reduction of P700 by following the increase of its absorbance at 832 nm (see "Experimental Procedures"). Photooxidation of P700 results in a broad absorbance in the near-IR due to the loss of the ground state character in oxidized chlorophyll and is monitored as an increase in absorbance at 832 nm. Optimization experiments indicated that the maximum amount of P700+ could be observed within 1 s of illumination with far-red light in the presence of sodium ascorbate and 2,6-dichlorophenolindophenol. The near-IR measuring beam is not able to photooxidize P700 by itself, thus eliminating a possibility of underestimating the amount of photochemically active P700. The maximum level of P700 oxidation is attained using far-red excitation, since this preferentially excites PS I, thereby decreasing the rate of P700+reduction in the light. In WT cells, P700 is oxidized within 200 ms of illumination and is completely rereduced within 2 s after termination of excitation (Fig. 3). We used continuous mass spectrometry to determine the in vivo rates of O2 evolution, CO2 fixation, and H2 photoevolution. We were able to distinguish photosynthetic from respiratory activities by the inclusion of [18O]O2 and [13C]CO2 (25Hanley J. Setif P. Bottin H. Lagoutte B. Biochemistry. 1996; 35: 8563-8571Crossref PubMed Scopus (42) Google Scholar). The disappearance of [13C]CO2 starting within 10 s after the commencement of illumination is indicative of the activation of the Calvin cycle enzymes, which then cooperate to convert gaseous CO2 to carbohydrate (30Hoober J.K. Siekevitz P. Chloroplasts: Cellular Organelles. Plenum Press, New York1984Crossref Google Scholar). The WT cells removed CO2 from the medium in a light-dependent manner at a steady-state rate of 1.5 μmol min−1 mg of chlorophyll−1 when illuminated with 300 μmol of photons m−2 s−1 (Fig. 4; TableI). When the photon flux was reduced 100-fold, CO2 uptake occurred at 1.2% of this rate (Fig.4). The O2 evolution rate was equivalent to this value (Table I), as expected; for each molecule of O2 produced, one molecule of CO2 is fixed. The respiration rate, as measured by uptake of 18O2, was not greatly affected by the light (Table I). Under anaerobic conditions, the chloroplast enzyme hydrogenase is induced and can serve as an alternate acceptor of electrons from ferredoxin (31Gaffron H. Rubin J. J. Gen. Physiol. 1942; 26: 219-240Crossref PubMed Scopus (466) Google Scholar, 32Stuart T.S. Gaffron H. Planta. 1972; 106: 91-100Crossref PubMed Scopus (27) Google Scholar, 33Stuart T.S. Gaffron H. Planta. 1972; 106: 101-112Crossref PubMed Scopus (44) Google Scholar). After removal of O2, production of H2 in WT cells was observed immediately upon illumination (Fig. 5).Table ISummary of characteristics of the strains under studyStrain genotypeAmount of PSIaLevels of PsaA and PsaD polypeptides in total cellular membranes were determined by densitometry of the immunoblot shown in Fig. 1, which is representative of results seen in other such experiments (data not shown). Levels of photooxidizable P700were determined by comparing the signals of the various mutants with WT as shown in Fig. 2. Note that the disagreement between the detection of PS I by immunological or spectroscopic means may be due to the different ways used to normalize the signals (i.e. to equal amounts of protein or to equal numbers of cells).Phototrophic growthbThe photoautotrophic rates are expressed qualitatively (+++, normal growth; ++, suboptimal growth; +, poor growth; −, no growth).CO2 uptake rate (light)cThese values are expressed as nmol min−1 (mg of chlorophyll)−1. The "light" rates were recorded at an illumination of 300 μmol of photons m−2 s−1, which allows fully saturated (low level) O2 photoevolution in PS I-deficient mutants and approximately 75% of the saturated value in WT cells. The lower limits of detection were 5 nmol of CO2min−1 (mg of chlorophyll)−1 and 1 nmol of H2min−1 (mg of chlorophyll)−1.O2 evolution rate (light)cThese values are expressed as nmol min−1 (mg of chlorophyll)−1. The "light" rates were recorded at an illumination of 300 μmol of photons m−2 s−1, which allows fully saturated (low level) O2 photoevolution in PS I-deficient mutants and approximately 75% of the saturated value in WT cells. The lower limits of detection were 5 nmol of CO2min−1 (mg of chlorophyll)−1 and 1 nmol of H2min−1 (mg of chlorophyll)−1.Respiration rate (dark)cThese values are expressed as nmol min−1 (mg of chlorophyll)−1. The "light" rates were recorded at an illumination of 300 μmol of photons m−2 s−1, which allows fully saturated (low level) O2 photoevolution in PS I-deficient mutants and approximately 75% of the saturated value in WT cells. The lower limits of detection were 5 nmol of CO2min−1 (mg of chlorophyll)−1 and 1 nmol of H2min−1 (mg of chlorophyll)−1.Respiration rate (light)cThese values are expressed as nmol min−1 (mg of chlorophyll)−1. The "light" rates were recorded at an illumination of 300 μmol of photons m−2 s−1, which allows fully saturated (low level) O2 photoevolution in PS I-deficient mutants and approximately 75% of the saturated value in WT cells. The lower limits of detection were 5 nmol of CO2min−1 (mg of chlorophyll)−1 and 1 nmol of H2min−1 (mg of chlorophyll)−1.H2 evolution rate (light)cThese values are expressed as nmol min−1 (mg of chlorophyll)−1. The "light" rates were recorded at an illumination of 300 μmol of photons m−2 s−1, which allows fully saturated (low level) O2 photoevolution in PS I-deficient mutants and approximately 75% of the saturated value in WT cells. The lower limits of detection were 5 nmol of CO2min−1 (mg of chlorophyll)−1 and 1 nmol of H2min−1 (mg of chlorophyll)−1.NuclearChloroplastPsaAPsaDP700%WTWT100100100+++150015303103001200WTpsaAΔ002−<5210500690<1WTpsaCΔ1821−<52102904853WTpsaB-FUD26575261dThe mutants FUD26 and B4-r2 show photoinduced signals with slightly slower oxidation and reduction kinetics.++10001070450600930F8WT10108+310330320320130F8psaAΔ201−<5140400550<1F8psaAΔ +psaA1084.3+280350450330150F8psaCΔ101−<5100310410<1B4WT703.3−<5130310420<1B4psaAΔ4203.6−<5120390540<1B4-r2WT15013039dThe mutants FUD26 and B4-r2 show photoinduced signals with slightly slower oxidation and reduction kinetics.++110016003205501100a Levels of PsaA and PsaD polypeptides in total cellular membranes were determined by densitometry of the immunoblot shown in Fig. 1, which is representative of results seen in other such experiments (data not shown). Levels of photooxidizable P700were determined by comparing the signals of the various mutants with WT as shown in Fig. 2. Note that the disagreement between the detection of PS I by immunological or spectroscopic means may be due to the different ways used to normalize the signals (i.e. to equal amounts of protein or to equal numbers of cells).b The photoautotrophic rates are expressed qualitatively (+++, normal growth; ++, suboptimal growth; +, poor growth; −, no growth).c These values are expressed as nmol min−1 (mg of chlorophyll)−1. The "light" rates were recorded at an illumination of 300 μmol of photons m−2 s−1, which allows fully saturated (low level) O2 photoevolution in PS I-deficient mutants and approximately 75% of the saturated value in WT cells. The lower limits of detection were 5 nmol of CO2min−1 (mg of chlorophyll)−1 and 1 nmol of H2min−1 (mg of chlorophyll)−1.d The mutants FUD26 and B4-r2 show photoinduced signals with slightly slower oxidation and reduction kinetics. Open table in a new tab Figure 5Photoproduction of H2 during dark/light transitions measured by mass spectrometry. Cultures were first subjected to anaerobic conditions in order to induce hydrogenase. The light (300 μmol of photons m−2s−1) was turned on when indicated by the arrow. Chlorophyll concentrations were 20 μg ml−1.A, WT (●) and psaAΔ (○) strains.B, F8 (●), F8-psaAΔ (○), and F8-psaAΔ+psaA (▾) strains. C, B4 (●) and B4-r2 (○) strains.View Large Image Figure ViewerDownload (PPT) In order to examine mutants that should contain no PS I, we made use of mutants with deletions in the chloroplast genes psaA, psaB, or psaC(Ref. 12Fischer N. Stampacchia O. Redding K. Rochaix J.D. Mol. Gen. Genet. 1996; 251: 373-380Crossref PubMed Scopus (142) Google Scholar; see Fig. 1). These deletions remove the entire coding sequence of psaA exon 3 (which encodes the last 661 amino acid residues of the 751-residue PsaA polypeptide), 96% of the psaB gene, or all of thepsaC gene. Thus, such deletion mutants should be incapable of reversion. We found that the psaA and psaC deletion mutants could not grow photoautotrophically and gave fluorescence induction patterns typical of mutants lacking PS I (Fig.2; results were identical forpsaBΔ, data not shown), in that the fluorescence rose to a high level and remained constant (34Takahashi Y. Goldschmidt-Clermont M. Soen S.Y. Franzen L.G. Rochaix J.D. EMBO J. 1991; 10: 2033-2040Crossref PubMed Scopus (163) Google Scholar). Photoautotrophic growth can be restored to these strains by reintroduction of the deleted gene (14Fischer N. Setif P. Rochaix J.D. Biochemistry. 1997; 36: 93-102Crossref PubMed Scopus (73) Google Scholar,15Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.-D. EMBO J. 1998; 17: 1-11Crossref PubMed Scopus (85) Google Scholar). We could detect no PsaA or PsaD polypeptides in membranes from the psaAΔ strain (Fig. 2 A). As has been observed previously (12Fischer N. Stampacchia O. Redding K. Rochaix J.D. Mol. Gen. Genet. 1996; 251: 373-380Crossref PubMed Scopus (142) Google Scholar, 34Takahashi Y. Goldschmidt-Clermont M. Soen S.Y. Franzen L.G. Rochaix J.D. EMBO J. 1991; 10: 2033-2040Crossref PubMed Scopus (163) Google Scholar), the psaCΔ mutant could accumulate some PsaA polypeptide at roughly 5–10% the WT value (Fig. 2 A). However, membranes from this mutant contained no detectable PsaD polypeptide, indicating that the stromal side of PS I is significantly perturbed due to the lack of PsaC. No photooxidizable P700could be detected in either mutant (Fig.3; Table I). We have previously shown that the psaBΔ mutant cannot fix CO2 (13Cournac L. Redding K. Bennoun P. Peltier G. FEBS Lett. 1997; 416: 65-68Crossref PubMed Scopus (19) Google Scholar). The psaAΔ and psaCΔmutants were also incapable of CO2 fixation (Fig.4; Table I), and no significant H2 photoevolution could be detected in thepsaAΔ mutant (Fig. 5; TableI). Significant O2 photoevolution does occur in these mutants (Table I); however, in this case, the electrons appear to be directed toward respiration rather than CO2 fixation or H2 evolution. Note that the light respiration rates in these PS I-deficient mutants are essentially the sum of the dark respiration and O2 evolution rates (Table I). Previous experiments (13Cournac L. Redding K. Bennoun P. Peltier G. FEBS Lett. 1997; 416: 65-68Crossref PubMed Scopus (19) Google Scholar) with the psaBΔ mutant under varying light showed that it could evolve O2 at a maximal rate about 4% that of the maximal WT rate but that this evolved O2 was almost quantitatively matched by an increase in the respiration rate (i.e. there is no net O2 evolution). The FUD26 mutant was shown to be PS I-deficient due to a 4-base pair deletion in the psaB gene, causing a frameshift and truncation of the last 10 kDa of the PsaB polypeptide (Ref. 19Girard-Bascou J. Choquet Y. Schneider M. Delosme M. Dron M. Curr. Genet. 1987; 12: 489-495Crossref PubMed Scopus (56) Google Scholar; see Fig. 1). Thus, this mutant should be incapable of synthesizing active PS I. However, we found that the FUD26 isolate provided to us was able to grow photoautotrophically (Fig.2 A) and had a fluorescence induction pattern consistent with significant reoxidation of the plastoquinone pool (data not shown). We detected the PsaA and PsaD polypeptides in significant amounts in membranes from this strain (Fig. 2 A). Moreover, we could detect photooxidizable P700 at a level approximately 60% that of WT (Fig. 3). This isolate was able to fix CO2 and photoevolve H2 at rates 67 and 77% of WT, respectively (Table I). We were surprised by the presence of PS I in this strain, considering the presence of a frameshift in the psaB gene. An isolate of FUD26 obtained from Dr. Girard-Bascou showed no growth on minimal medium, similar to the original description of this mutant (20Girard-Bascou J. Curr. Genet. 1987; 12: 483-488Crossref Scopus (25) Google Scholar). We hypothesize that the mutant has undergone some type of mutation event that allows it to synthesize some full-length PsaB. We note that Greenbaum et al. (35.Greenbaum, E., Lee, J. W., Proceedings of the 1998 Hydrogen Program Annual Review, 1998, 1, 7, Office of Scientific and Technical Information, Alexandria, VA.Google Scholar) estimated during their recent experiments that this mutant had levels of PS I equivalent to 11–14% of WT; it would thus appear that this mutant can revert to various levels of PsaB expression. These results highlight the danger of using mutants in which the genetic information specifying the protein of interest has not been completely obliterated. The F8 mutant has a nuclear mutation that preventstrans-splicing of the psaA mRNA (Refs. 10Girard J. Chua N.H. Bennoun P. Schmidt G. Delosme M. Curr. Genet. 1980; 2: 215-221Crossref PubMed Scopus (65) Google Scholar and11Goldschmidt-Clermont M. Girard-Bascou J. Choquet Y. Rochaix J.D. Mol. Gen. Genet. 1990; 223: 417-425Crossref PubMed Scopus (115) Google Scholar; see Fig. 1). The F8 strain was able to grow slowly on minimal medium (Fig. 2 A) and exhibited a fluorescence induction pattern characteristic of mutants containing small amounts of PS I (Fig. 2 B; Refs. 15Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.-D. EMBO J. 1998; 17: 1-11Crossref PubMed Scopus (85) Google Scholar, 36Bennoun P. Girard J. Chua N.-H. Mol. Gen. Genet. 1977; 153: 343-348Crossref Scopus (25) Google Scholar). Membranes from this strain contained small but detectable amounts of the PsaA and PsaD subunits. Using densitometry, we estimated that these polypeptides were present at roughly 10% of the WT amount when normalized to total membrane protein (note that PsaA and PsaD were present at the same stoichiometry as in the WT). Photooxidizable P700 in this strain represented roughly 8% of the WT level (Fig. 3; Table I), indicating that a small amount of active PS I was present in this strain. We observed light-dependent CO2 fixation at roughly 20% of the WT rate (Fig. 4; Table I) and H2photoproduction at roughly 10% the WT rate (Fig. 5; Table I). An isolate of F8 from the Chlamydomonas Stock Center was similar to the one shown here in that it contained small amounts of PsaA and grew slowly on minimal medium (data not shown); thus, this phenotype might be a general phenomenon and not due to an isolated reversion event.