Jeannine Maroc

Oligomeric form of the light-harvesting chlorophyll a + b-protein complex CP II, phosphatidyldiacylglycerol, Δ3-trans-hexadecenoic acid and energy transfer in Chlamydomonas reinhardtii, wild type and mutants

Abstract Analyses of chlorophyll-protein complexes and of lipids were performed with the wild type of Chlamydomonas reinhardtii and three non-photosynthetic mutants: Fl 39, which was a ‘classical’ high-fluorescent Photosystem II (PS II)-lacking mutant, and mf 1 and mf 2, which lacked also functional PS II but were low-fluorescent and showed an abnormally predominant energy transfer from the main light-harvesting antenna towards Photosystem I. An oligomeric form of the chlorophyll a + b -protein complex CP II was clearly isolated from the wild type and the mutant Fl 39 but it was not detected in the mutants mf 1 and mf 2. The three mutants showed total lipid contents close to or greater than that of the wild type. Their phosphatidyldiacylglycerol (PG) contents, on a chlorophyll basis, were higher ( Fl 39) or 1.4- ( mf 1) and 2.0- ( mf 2) times lower than that of the wild type. The fatty acid compositions of the wild type and of the mutant Fl 39 were comparable, showing about equal amounts of a C18 series and a C16 series which included the Δ3- trans -hexadecenoic acid (C16:1- trans ). This C16:1- trans was not detected in the mutants mf 1 and mf 2 which contained the other fatty acids. These results indicate correlations between lack of C16:1- trans -containing PG, lack of an oligomeric form of CP II and an impaired mechanism of the regulation of excitation energy transfer from the main chlorophyll a + b antenna.

Low-temperature fluorescence emission spectra and chlorophyll-protein complexes in mutants of Chlamydomonas reinhardtii: Evidence for a new chlorophyll-a-protein complex related to Photosystem I

With the aim of confirming the relationships between low-temperature emission bands of chlorophyll fluorescence of whole cells and chlorophyll-protein complexes as defined by their electrophoretic behaviour, a study was performed with the wild type of Chlamydomonas reinhardtii and eight mutants which lacked one or several chlorophyll-protein complexes and showed impaired photosynthetic functions. Chlorophyll-protein complexes from chloroplast fragments or Triton X-100-treated particles, which were enriched in both Photosystems I and II (PS I and PS II), were analyzed by lithium dodecylsulfate polyacrylamide gel electrophoresis at 4°C, using either lithium dodecylsulfate or n-octyl-β-d-glucopyranoside in the solubilization mixtures. Absorption and fluorescence emission spectra were measured at 77 K with whole cells and also, for fluorescence, with isolated chlorophyll-protein complexes. Fluorescence emissions in the 680–682 nm (F682), 686 nm (F686), 696 nm (F696), 703 nm (F703), 707 nm (F707) and 712–717 nm (F715) regions were observed with whole cells of the different strains. A comparative study of the chlorophyll-protein complexes, the fluorescence emissions and the photochemical activities shown by each strain confirmed the following correlations, which have been previously described or proposed in the literature for algae or higher plants: F682 with CP II, the chlorophyll (Chl) a + b complex corresponding to the main light-harvesting antenna; F686 and F696 with CP IV and CP III, the Chl a complexes corresponding to the antenna and to the core of PS II respectively; F707 with CP 0, the Chl a + b complex part of the PS I antenna; F715 with CP I, the Chl a complex corresponding to the core of PS I. A new PS-I-related Chl a-protein complex, tentatively designated CP 0a, was also observed. This complex showed an apparent relative molecular mass slightly smaller than that of CP I. It was the only PS I-related complex in the double mutant Fl 5 Pg 27 which lacks Chl b, CP 0, CP I and CP II. It appeared to be correlated with F703, which was observed with cells of the same double mutant. The emission spectrum of another mutant, Fl 50 which contained CP I and CP II but was deficient in CP 0 and CP 0a, showed a F715 contribution significantly reduced, indicating that CP 0 and CP 0a play an essential part in energy transfer from CP II to CP I. The probable pathways of light energy transfer in C. reinhardtii were examined. It is proposed that CP 0a acts as a connecting antenna between CP 0 and CP I, the energy transfer from CP II to PS I occurring through the following sequence: CP II → CP 0 → CP 0a → CP I core antenna → PS I reaction center.

Gel electrophoresis of chloroplast membranes of mutants of Chlamydomonas reinhardii which have impaired Photosystem II function and lack photosynthetic cytochromes

Abstract Photosystem (PS) II-enriched particles or chloroplast fragments of the wild type and of three nonphotosynthetic mutants of Chlamydomonas reinhardii , which lack chloroplast cytochromes, were analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C to locate which chlorophyll complexes and which proteins are associated with cytochrome b -559. Two mutants, Fl 39 and Fl 50, have previously been shown to contain, respectively, 3.6- and 2.7-times less hydroquinone-reducible high-potential cytochrome b -559 than the wild type. They have impaired PS II functions. In the presence of ADRY agents: carbonyl cyanide p -trifluoromethoxyphenylhydrazone (FCCP), 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) or 2-(3,4,5-trichloro)-anilino-3,5-dinitrothiophene (ANT 2s), Fl 50 carried out photo-oxidation of cytochrome b -559 with half the amplitude of that of the wild type. No photo-oxidation was observed with Fl 39. We show here that in both these mutants chlorophyll-protein complexes CP III, CP IV and CP V were missing. There were traces of the corresponding apoproteins (45 000, 42 000 and 33 000 daltons, respectively) in Fl 50 but none in Fl 39. In addition, a 19 000 dalton protein was missing in Fl 39 and present in a very small amount in Fl 50. In another mutant, Fl 9, previously characterized as lacking both cytochromes b -563 and c -553 with a normal cytochrome b -559 content, CP III-CP V and the 19 000 dalton protein were detected. CP I (110 000 daltons) and CP II (24 000 daltons) were present in all strains. These observations confirmed the close relationship between deficiencies in cytochrome b -559, lack of CP III and CP IV and anomalies in the photochemistry of PS II. They provided additional evidence that CP V and a 19 000 dalton protein are also involved in this PS II photochemistry. Staining of the gels with 3,3′,5,5′-tetramethylbenzidine and H 2 O 2 allowed us to distinguish clearly four heme protein bands having peroxidase activity. Three of these bands (45 000, 42 000 and 19 000 daltons), which were shown in wild-type, Fl 39 and Fl 50 preparations but not in Fl 9, appeared related to cytochromes b -563 and c -553. The fourth heme protein (14 000 daltons) occurred in wild type and Fl 9 but was missing in Fl 39 and Fl 50; it appeared related to cytochrome b -559.

Photooxidation of the cytochrome b-559 in the presence of various substituted 2-anilinothiophenes and of some other compounds, in Chlamydomonas reinhardtii.

Five substituted 2-anilinothiophenes and two substituted carbonylcyanide-phenylhydrazones were comparatively studied with respect to their capacities for inducing photooxidation of the cytochrome b-559 in chloroplast fragments and in whole cells of Chlamydomonas reinhardtii (wild type and P-700-lacking mutant Fl 5). In addition, some other compounds: antimycin A, picric acid, tetraphenylboron and NH4Cl were also tested. Cytochrome b-559 photooxidations were clearly observed in the presence of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), 2-(3,4,5-trichloro)anilino-3,5-dinitrothiophene (ANT 2s), 2-(4-chloro)anilino-3,5-dinitrothiophene and, with greater amplitudes, in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone and carbonylcyanide-m-chlorophenylhydrazone, both in whole cells and in chloroplast fragments. Picric acid, antimycin A and tetraphenylboron were also able to induce cytochrome b-559 photooxidation in chloroplast fragments, but not in whole cells. In the wild type, the highest photoinduced redox changes were 1.1 (carbonylcyanide-p-trifluoromethoxyphenylhydrazone, carbonylcyanide-m-chlorophenyl-hydrazone) and 0.6 (ANT 2p, ANT 2s) mumol of oxidized cytochrome b-559/1 mmol of chlorophyll, corresponding to 40% and 23% of the redox changes which could be induced chemically. All these cytochrome b-559 photooxidations, the greater part of which was inhibited by 3-(3,4-dichloropheny)-1,1-dimethylurea and occurred in the mutant Fl 5, appeared to be mainly Photosystem II-dependent reactions. But 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive Photosystem I-dependent photooxidations of cytochrome b-559 occurred also in the wild type. On the other hand, 2-(4-dimethylamine)-anilino-3,5-dinitrothiophene, 2-N-methyl-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene and NH4Cl did not induce any cytochrome b-559 photooxidation. These results were discussed taking in consideration the nature of the molecular substitutions of the various tested substances and their respective acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis capacities which had been defined elsewhere by Renger (Renger, G. (1972) Biochim. Biophys. Acta 256, 428-439) for spinach chloroplasts. Like the acceleration of the deactivation reactions of the water-splitting enzyme system Y effect, the capacity for inducing the cytochrome b-559 photooxidation appeared dependent on the acidity of the NH group and on the number of halogenous substituents in the aromatic ring of the molecule. The greatest action towards cytochrome b-559 photooxidation was obtained with the most active acceleration of the deactivation reactions of the water-splitting enzyme system Y agents: carbonylcyanide-p-trifluoromethoxyphenylhydrazone, ANT 2p and ANT 2s.

Étude des échanges de CO2 des cellules entières et des activités photochimiques des fragments de chloroplastes de trois mutants non photosynthétiques de Chlamydomonas reinhardti

Abstract Studies of the CO 2 exchanges of whole cells and of the photochemical activities of chloroplast fragments, with three non-photosynthetic mutants of Chlamydomonas reinhardti Investigations were performed in order to determine the different kinds of electron transport, which are carried out by three non-photosynthetic mutants ( Fl 5, Fl 9, Fl 15) of Chlamydomonas reinhardti . These mutants had been previously selected for their fluorescence and O 2 evolution anomalies, which indicate a blocking of the internal electron transport chain close to Photoreaction II. Aerobic and anaerobic (with H 2 or N 2 ) CO 2 exchanges of whole cells were measured with a pCO 2 electrode. Photochemical activities of chloroplast fragments with various exogenous electron carriers were measured spectrophotometrically. The three mutants were unable to fix CO 2 in the light, in air and in the presence of H 2 with 3- p -chlorophenyl-1,1-dimethylurea (CMU) only; in addition, they could not perform photoreduction of NADP + with water. However, Fl 9 and Fl 15 were able to carry out photoreduction of NADP + with 2,6-dichlorophenolindophenol (DCIP)-ascorbate, photooxidation of exogenous cytochrome c , and CO 2 fixation in the light in the presence of H 2 , CMU and phenazine methosulfate. Fl 5 did not perform any of these reactions. With DCIP and plastocyanin the Hill activities of the three mutants were very weak, compared to those of the wild type; with K 3 Fe(CN) 6 , the activities were relatively more important, particularly for Fl 5. These results indicate a non-functioning of System I in Fl 5, on the one hand, and a blocking of the electron transport chain between the two photoreactions (both of which are functional) in Fl 9 and in Fl 15, on the other. An interpretation is attempted, taking into consideration the analytical results obtained elsewhere (lack of P700 for Fl 5, deficiencies in bound cytochromes c -553 and b -563 for Fl 9 and Fl 15). Some points, concerning the nature of the defect(s) in each mutant, the reaction sites of the various cytochromes, and the possibility of different pathways for the photosynthetic electron transports, in vivo and in vitro , are discussed.

Recherche de plusieurs transporteurs d'électrons, notamment des cytochromes b-559 et c-553, chez trois mutants non photosynthétiques de Chlamydomonas reinhardi.

Abstract Studies of several electron carriers, particularly cytochromes b -559 and c -553 in three non-photosynthetic mutants of Chlamydomonas reinhardi Biochemical and spectroscopic analyses were performed in order to investigate the electron-carrier contents of three non-photosynthetic mutants ( Fl 5, Fl 9 and Fl 15) of Chlamydomonas reinhardi . These mutants exhibit several physiological amomalies, which indicate a blocking of the internal electron transport close to Photoreaction II. Quinones and soluble proteins were analysed by chromatography. With the insoluble cell debris, difference absorption spectra were measured at 20° and at −190°. The plastoquinone A, soluble cytochrome c -553, plastocyanin, ferredoxin and ferredoxin-NADP + reductase contents of each mutant and of the wild type were comparable. Cytochrome b -559, also, was present in the four strains. But it appeared that the mutants Fl 9 and Fl 15 showed only traces of the insoluble (bound) form of cytochrome c -553; this insoluble form was present, in appreciable amounts, in the wild type and in the mutant Fl 5. The wild type contained about: I molecule of cytochrome b -559 per 130 molecules of chlorophyll ( a + b ) and I molecule of bound cytochrome c -553 per 500 molecules of chlorophyll. It seems probable that the mutants are affected by structural defects, wich may be of a different nature for Fl 9 and Fl 15, on the one hand, and for Fl 5 on the other. However, the possibility of the lack of an unknown electron carrier in these mutants cannot be ruled out.

Chlorophyll a′ and pheophytin a, as determined by HPLC, in photosynthesis mutants and double mutants of Chlamydomonas reinhardtii

Abstract HPLC analysis of chlorophyll (Chl) a ′ and pheophytin (Pheo) a content was run for wild type Chlamydomonas reinhardtii and seven mutants with impaired photosynthetic functions. Four of them had no or little Photosystem II (PS II) activity. Two had no Photosystem I (PS I) activity and no P-700. In the seventh mutant, both photosystems were present but the main light-harvesting antenna (the chlorophyll-protein complex corresponding to PS II: CP II) was totally lacking because of the absence of Chl b . In mutants impaired in PS II activity, Pheo a is either lacking or found in only low amounts in direct relation with the loss in PS II activity. These results are in agreement with the well-known role of Pheo a as a primary electron acceptor in PS II. In mutants impaired in PS II activity the Chl a ′ level is normal. In mutants impaired in PS I activity (P-700 less mutants), only half of the normal content of Chl a ′ is present. So Chl a ′ is associated exclusively with PS I as demonstrated before by Watanabe (Watanabe, T., Kobayashi, M., Hongu, A., Nakazato, M., Hiyama, T. and Murata, N. (1985) FEBS Lett. 191, 252–256). But only half the Chl a ′ content could be P-700. This last result contradicts Watanabes conclusion that the whole Chl a ′ constitutes P-700. On the other hand, there is no relation between the presence of Chl a ′ and the second Chl a -protein complex of PS I: CP 0a. The possible function of Chl a ′ related to PS I activity is discussed.

Recherche du cytochrome b-563 et du p700 chez trois mutants non photosynthétiques de Chlamydomonas reinhardti

Abstract Studies of cytochrome b-563 and P 700 in three non-photosynthetic mutants of Chlamydomonas reinhard An investigation into the presence of cytochrome b -563 and of P700 in three non-photosynthetic mutants ( Fl 5, Fl 9, Fl 15) of Chlamydomonas reinhardti was carried out. These three mutants exhibit several functional anomalies (described elsewhere), which indicate that the electron transport chain between the two photoreactions is blocked. In addition, Fl 5 is unable to carry out any reaction related to System I. Mutants Fl 9 and Fl 15 had less than 19% of the cytochrome b -563 content found in the wild type (which was about 0.27 mole per 100 moles chlorophyll); mutant Fl 5 had more than 80% of this content. The deficiencies (only traces) in bound cytochrome c -553, previously observed with mutants Fl 9 and Fl 15, but not Fl 5, were confirmed (in the wild type, there is about 0.20 mole bound cytochrome per 100 moles chlorophyll). Photosystem I particles, prepared from wild type and mutants Fl 9 and Fl 15 chloroplast fragments, had about 2 ( Fl 9, Fl 15) and 3 (wild type) moles P700 per 100 moles chlorophyll. Mutant Fl 5 particles showed neither P700 spectroscopic characteristics nor photooxidation activity; their chlorophyll a/b ratio was lower by a factor of 2 and protein/chlorophyll ratio about 8 times higher than in the wild type particles. This mutant appears to lack P700.

Chlorophyll—protein complexes related to photosystem I in Chlamydomonas reinhardtii

Abstract A study of the photosystem I (PS I)-related chlorophyll (Chl)—protein complexes of the unicellular green alga Chlamydomonas reinhardtii was performed, using the wild type and six photosynthesis mutants. Three complexes have been previously described in the literature: CP Ia, undissociated PS I complex; CP 0, Chl a + b peripheral antenna of PS I; CP I, Chl a complex corresponding to the PS I reaction center and its core antenna. Another Chl a complex, CP 0a, has been more recently described by us. The electrophoretic behaviors of these complexes and of their polypeptide subunits have been studied using different solubilization mixtures and lithium dodecylsulfate polyacrylamide gels. It was determined that the non-denatured complex CP 0a shows an apparent relative molecular mass ( M r in the 80 000 range and that two polypeptides of M r = 50 000 and 38 000 resulted from its dissociation. Low temperature fluorescence emission spectra of cells of the wild type and of three mutants were measured both in oxidized and reduced plastoquinone pool conditions. It appeared obvious that, in a CP 0a-lacking mutant, the excitation energy transfer from the main light-harvesting antenna towards PS I was impaired and inefficient. This is interpreted as indicating that CP 0a plays the essential role of a connecting antenna between the peripheral antenna CP 0 and the core antenna (CP I) of PS I.

La photooxydation du cytochrome b-559, en presence de carbonylcyanure-p-trifluorométhoxyphenylhydrazone et de 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, ou de p-benzoquinone, chez trois mutants non-photosynthétiques de Chlamydomonas reinhardti

Cytochrome b-559 photooxidation in the presence of carbonyl cyanide p-trifluorometh-oxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or p-benzoquinone in three non-photosynthetic mutants of Chlamydomonas reinhardti Studies of absorbance changes related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3-p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563. In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 μmol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl 5) or one fifth (Fl 9, Fl 15) of the total amount of this cytochrome. Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553. A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also. An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.