Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

The carboxy-terminal extension of the D1-precursor protein is dispensable for a functional photosystem II complex in Chlamydomonas reinhardtii

The D1-precursor protein of the photosystem II reaction centre contains a carboxy-terminal extension whose proteolytic removal is necessary for oxygen-evolving activity. To address the question of the role of the carboxy-terminal extension in the green alga Chlamydomonas reinhardtii, we truncated D1 by converting codon Ser345 of the psbA gene into a stop codon. Particle gun transformation of an in vitro modified psbA gene fragment also carrying mutations conferring herbicide resistance yielded a homoplasmic transformant containing the stop codon. Since oxygen evolution capacity is not affected in this mutant as compared with herbicide-resistant control cells, the carboxy-terminal extension is dispensable for a functional photosystem II complex under normal growth conditions.     

The cytoplasmic ribosomes of Chlamydomonas reinhardtii: characterization of antibiotic sensitivity and cycloheximide-resistant mutants

Summary In vitro protein synthesis was used to characterize the antibiotic sensitivity of cytoplasmic ribosomes from wild-type and antibiotic-resistant strains of Chlamydomonas reinhardtii. Cytoplasmic ribosomes from two cycloheximide-resistant mutants, act-1 and act-2, were resistant to the antibiotic in vitro. The alteration effected by the act-1 mutation, which was dominant in diploids, was localized to the large subunit of the cytoplasmic ribosomes, but no ribosomal protein alterations were detected using two-dimensional gel electrophoresis. The act-2 mutation, which was semidominant in diploids, was frequently associated with a charge alteration in the large subunit ribosomal protein (r-protein) cyL38 that segregated independently from the antibiotic-resistant phenotype in crosses.     

Isolation and characterization of cDNA clones encoding photosystem I subunits with molecular masses 11.0, 10.0 and 8.4 kDa from Chlamydomonas reinhardtii

Summary cDNA clones encoding three photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses 13, 5 and 3 kDa (thylakoid polypeptides 28, 35 and 37; P28, P35 and P37, respectively) were isolated using gene specific oligonucleotides as probes. The sequences of these oligonucleotides were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that the proteins are encoded by single-copy genes. The mRNA sizes of the three components are 960 (P28), 1120 (P35) and 790 (P37) nucleotides. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the nascent polypeptides possess N-terminal transit sequences that are removed to give mature proteins of 11.0 (P28), 10.0 (P35) and 8.4 (P37) kDa. Analysis of the deduced protein sequences suggests that P28 and P35 are extrinsic membrane proteins and that P37 spans the thylakoid membrane. All three proteins have short transit peptides that probably route them to the stromal side of the thylakoid membrane.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - RuBisCO ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P28, P35 and P37 Chlamydomonas reinhardtii thylakoid polypeptides 28, 35 and 37 The nucleotide sequences presented here will appear in the EMBL/Genbank/DDBJ Nucleotide Sequence Databases under the accession numbers X15164 (11.0 kDa subunit; P28), X15165 (10.0 kDa subunit; P35) and X15166 (8.4 kDa subunit; P37)     

Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O₂ evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111–1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.     

The psbO mutant of Chlamydomonas reinhardtii is capable of assembling stable, photochemically active reaction center of photosystem II

The functional status of photosystem II (PSII) complex in the dark-grown PsbO-deficient mutant of green alga Chlamydomonas reinhardtii was studied. It was found that ΔpsbO mutant cells of C. reinhardtii grown under heterotrophic conditions (dark + acetate) were capable of assembling stable, photochemically-competent reaction centers of PSII (as confirmed by immunological analysis of D1 protein level, pigments content and photoinduced changes of PSII chlorophyll fluorescence yield), while O₂-evolution activity was not revealed. The ratio F _v/F _m for the dark-grown ΔpsbO mutant C. reinhardtii was 0.37 and that for the dark-grown wild type cells was 0.56. Analysis of chlorophyll fluorescence induction curve indicated that the absence of oxygen-evolving activity could be due to some defects in the organization of the PSII catalytic manganese cluster. Decrease of the rate of the electron donation from water-oxidizing complex to the PSII reaction center as well as the appearance of an additional transient fluorescence peak during the dark relaxation of F _v testify to the damages to the PSII donor side. The data obtained suggest that the dark-grown PsbO-deficient cells of C. reinhardtii are able to form stable, photochemically active PSII reaction center, unable to oxidize water due to probable defects in the assembly of the manganese cluster.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Role of singlet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii

High light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II and causes photooxidative stress. In Chlamydomonas reinhardtii, singlet oxygen also induces the expression of the nuclear-encoded glutathione peroxidase homologous gene GPXH. We provide evidence that singlet oxygen stimulates GPXH expression by activating a signaling mechanism outside the thylakoid membrane. Singlet oxygen from photosystem II could be detected with specific probes in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells. This indicates that singlet oxygen can stimulate a response farther from its production site than generally believed.     

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

We have developed a rapid method for isolation of the Photosystem I (PS1) complex from Chlamydomonas reinhardtii using epitope tagging. Six histidine residues were genetically added to the N-terminus of the PsaA core subunit of PS1. The His₆-tagged PS1 could be purified with a yield of 80–90% from detergent-solubilized thylakoid membranes within 3 h in a single step using a Ni-nitrilotriacetic acid (Ni-NTA) column. Immunoblots and low-temperature fluorescence analysis indicated that the His₆-tagged PS1 preparation was highly pure and extremely low in uncoupled pigments. Moreover, the introduced tag appeared to have no adverse effect upon PS1 structure/function, as judged by photochemical assays and EPR spectroscopy of isolated particles, as well as photosynthetic growth tests of the tagged strain. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The herbicide-resistant D1 mutant L275F of Chlamydomonas reinhardtii fails to show the bicarbonate-reversible formate effect on chlorophyll a fluorescence transients

Herbicide-resistant mutants of the eukaryotic green alga Chlamydomonas reinhardtii, that are altered in specific amino acids in their D-1 protein, show differential bicarbonate-reversible formate effects. These results suggest the involvement of D1 protein in the bicarbonate effect. A 25 mM formate treatment of mixotrophically or photoautotrophically grown wild type cells results in a slower rise of chlorophyll a fluorescence transient followed by a dramatically slowed decline during measurements in continuous light. These effects are fully reversed upon addition of 10 mM bicarbonate. The mutant BR-202 [L275F] is, however, highly insensitive to 25 mM formate suggesting that a significant change in formate (bicarbonate) binding has occurred in helix V of the D1 protein near histidine involved in Fe binding. With the exception of DCMU-4 [S264A], which is considerably more sensitive to formate than the wild type, five other different [V219I, A25IV, F255Y, G256D and cell-wall deficient CW-15] mutants display a relatively similar response to formate as wild type. Absence of formate effect on a PS II-lacking [FuD-7] mutant confirms the sole involvement of PS II in the bicarbonate effect.     

Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.     

Changes in Photosystem II fluorescence in Chlamydomonas reinhardtii exposed to increasing levels of irradiance in relationship to the photosynthetic response to light

The effects of a 60 min exposure to photosynthetic photon flux densities ranging from 300 to 2200 mol m^–2s^–1 on the photosynthetic light response curve and on PS II heterogeneity as reflected in chlorophyll a fluorescence were investigated using the unicellular green alga Chlamydomonas reinhardtii. It was established that exposure to high light acts at three different regulatory or inhibitory levels; 1) regulation occurs from 300 to 780 mol m^–2s^–1 where total amount of PS II centers and the shape of the light response curve is not significantly changed, 2) a first photoinhibitory range above 780 up to 1600 mol m^–2s^–1 where a progressive inhibition of the quantum yield and the rate of bending (convexity) of the light response curve can be related to the loss of Q_B-reducing centers and 3) a second photoinhibitory range above 1600 mol m^–2s^–1 where the rate of light saturated photosynthesis also decreases and convexity reaches zero. This was related to a particularly large decrease in PS II centers and a large increase in spill-over in energy to PS I.Abbreviations Chl chlorophyll - DCMU 3,(3,4-dichlorophenyl)-1,1-dimethylurea - F_M maximal fluorescence yield - F_pl intermediate fluorescence yield plateau level - F₀ non-variable fluorescence yield - F_v total variable fluorescence yield (F_M-F₀) - initial slope to the light response curve, used as an estimate of initial quantum yield - convexity (rate of bending) of the light response curve of photosynthesis - LHC light-harvesting complex - P_max maximum rate of photosynthesis - PQ plastoquinone - Q photosynthetically active photon flux density (400–700 nm, mol m^–2s^–1) - PS photosystem - Q_A and Q_B primary and secondary quinone electron acceptor of PS II     

CDKA and CDKB kinases from Chlamydomonas reinhardtii are able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae

Summary. Cyclin-dependent kinases (CDK) play a key role in coordinating cell division in all eukaryotes. We investigated the capability of cyclin-dependent kinases CDKA and CDKB from the green alga Chlamydomonas reinhardtii to complement a Saccharomyces cerevisiae cdc28 temperature-sensitive mutant. The full-length coding regions of algal CDKA and CDKB cDNA were amplified by RT-PCR and cloned into the yeast expression vector pYES-DEST52, yielding pYD52-CDKA and pYD52-CDKB. The S. cerevisiae cdc28-1N strain transformed with these constructs exhibited growth at 36 °C in inducing (galactose) medium, but not in repressing (glucose) medium. Microscopic observation showed that the complemented cells had the irregular cylindrical shape typical for G₂ phase-arrested cells when grown on glucose at 36 °C, but appeared as normal budded cells when grown on galactose at 36 °C. Sequence analysis and complementation tests proved that both CDKA and CDKB are functional CDC28/cdc2 homologs in C. reinhardtii. The complementation of the mitotic phenotype of the S. cerevisiae cdc28-1N mutant suggests a mitotic role for both of the kinases. Correspondence: K. Bišová, Laboratory of Cell Cycles of Algae, Institute of Microbiology, Academy of Sciences of the Czech Republic, 379 81 Třeboň, Czech Republic.     

Fluoroacetamide resistance mutations in Chlamydomonas reinhardtii

Acetamide, a nitrogen and carbon source for Chlamydomonas reinhardtii, is hydrolyzed by acetamidase to ammonium and acetate. It also induces urea pathway activities. Fluoroacetamide (F-acetamide) is toxic to wild-type through conversion to F-citrate, a respiratory inhibitor. Resistant mutants were selected on plates of F-acetamide plus urea. When tested on acetamide plates two mutant classes were obtained, acm⁺ (utilized acetamide as sole N source) and acm^-. All acm⁺ isolates had acetamidase activity and were obligate phototrophs (i.e. dark-diers). Acm^- isolates had either normal urea assimilation (ure⁺) or lacked all urea pathway activities, namely transport, urea carboxylase and allophanate hydrolase (ure^-). Inheritance patterns for both types indicated single nuclear gene mutations. The acm^- ure⁺ type presumably resulted from a defective acetamidase gene, and the acm^- ure^- strains might be regulatory gene mutants. Temperature conditional F-acetamide tolerant mutants were also obtained. Acetamidase extracted from one such strain was more thermolabile than the wild-type enzyme, indicating a mutation in the coding region. The hypothesis that acetamidase is involved in urea assimilation was not supported by the genetic and biochemical evidence.Abbreviations F-acetamide fluoroacetamide - F-acetate fluoroacetate - TAP tris-acetate-phosphate medium - CDB Chlamydomonas dilution buffer - TCA trichloroacetic acid - AH allophanate hydrolase - UC urea carboxylase - PAR photosynthetically active radiation - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Temperature-dependent adjustment of the thermal stability of photosystem II in vivo: possible involvement of xanthophyll-cycle pigments

Moderately elevated temperatures induce a rapid increase in the heat and light resistance of photosystem II (PSII) in higher-plant leaves. This phenomenon was studied in intact potato leaves exposed to 35 °C for 2 h, using chlorophyll fluorometry, kinetic and difference spectrophotometry and photoacoustics. The 35 °C treatment was observed to cause energetic uncoupling between carotenoids and chlorophylls: (i) the steady-state chlorophyll fluorescence emission excited by a blue light beam (490 nm) was noticeably reduced as compared to fluorescence elicited by orange light (590 nm) and (ii) the quantum yield for photosynthetic oxygen evolution in blue light (400–500 nm) was preferentially reduced relative to the quantum yield measured in red light (590–710 nm). Analysis of the chlorophyll-fluorescence and light-absorption characteristics of the heated leaves showed numerous analogies with the fluorescence and absorption changes associated with the light-induced xanthophyll cycle activity, indicating that the carotenoid species involved in the heat-induced pigment uncoupling could be the xanthophyll violaxanthin. More precisely, the 35 °C treatment was observed to accelerate and amplify the non-photochemical quenching of chlorophyll fluorescence (in both moderate red light and strong white light) and to cause an increase in leaf absorbance in the blue-green spectral region near 520 nm, as do strong light treatments which induce the massive conversion of violaxanthin to zeaxanthin. Interestingly, short exposure of potato leaves to strong light also provoked a significant increase in the stability of PSII to heat stress. It was also observed that photosynthetic electron transport was considerably more inhibited by chilling temperatures in 35 °C-treated leaves than in untreated leaves. Further, pre-exposure of potato leaves to 35 °C markedly increased the amplitude and the rate of light-induced changes in leaf absorbance at 505 nm (indicative of xanthophyll cycle activity), suggesting the possibility that moderately elevated temperature increased the accessibility of violaxanthin to the membrane-located de-epoxidase. This was supported by the quantitative analysis of the xanthophyll-cycle pigments before and after the 35 °C treatment, showing light-independent accumulation of zeaxanthin during mild heat stress. Based on these results, we propose that the rapid adjustment of the heat resistance of PSII may involve a modification of the interaction between violaxanthin and the light-harvesting complexes of PSII. As a consequence, the thermoresistance of PSII could be enhanced either directly through a conformational change of PSII or indirectly via a carotenoid-dependent modulation of membrane lipid fluidity.Abbreviations and Symbols Fo and Fm initial and maximal level of chlorophyll fluorescence, respectively - Fv = Fm — Fo variable chlorophyll fluorescence - LHC(II) light-harvesting chlorophylla/b-protein complexes (of PSII) - photoacoustically measured quantum yield of photosynthetic oxygen evolution (in relative values) - _P fluorimetrically measured quantum yield of PSII photochemistry in the light - PFD photon flux density - q_E pH dependent quenching of chlorophyll fluorescence We thank Dr. J-L Montillet (CEA-Cadarache) for the use of his HPLC apparatus and Professor Y. Lemoine (University of Lille, France) for technical advice on HPLC.     

Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii

Chlorophyll (Chl) a fluorescence transient, a sensitive and non-invasive probe of the kinetics and heterogeneity of the filling up of the electron acceptor pool of Photosystem II (PS II), was used to characterize D1-mutants of Chlamydomonas reinhardtii. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which provides the first measured data point at 10 s and allows data accumulation over several orders of magnitude of time, we have characterized, for the first time, complete Chl a fluorescence transients of wild type (WT), cell wall less (CW-15) C. reinhardtii and several herbicide-resistant mutants of the D1 proteins: D1-V219I A251V, F255Y, S264A G256D and L275F. In all cases, the Chl a fluorescence induction transients follow a pattern of O-J-I-P where J and I appear as two steps between the minimum Fo (O) and the maximum Fmax (Fm, P) levels. The differences among the mutants are in the kinetics of the filling up of the electron acceptor pool of PS II (this paper) in addition to those in the re-oxidation kinetics of Q_A ^– to Q_A, published elsewhere (Govindjee et al. (1992) Biochim Biophys. Acta: 1101: 353–358; Strasser et al. (1992) Archs. Sci. Genève 42: 207–224) and not in the ratio of the maximal fluorescence Fm to the initial fluorescence Fo. The value of this experimental ratio is Fm/Fo = 4.4±0.21 independent of the mutation. At 600 W m^–2 of 650 nm excitation, distinct hierarchy in the fraction of variable Chl a fluorescence at the J level is observed: S264A > A251V G256A > L275F V219I > F255Y CW-15 WT. At 300 and 60 W m^–2 excitation, a somewhat similar hierarchy among the mutants was observed for the intermediate levels J and I. Addition of bicarbonate-reversible inhibitor formate did not change the O to J phases, slowed the I to P rise, and in many cases, slowed the decay of fluorescence beyond the P level. These observations are interpreted in terms of formate effect being on the acceptor rather than on the donor side (S-states) of PS II. The formate effect was different in different mutants, with L275F being the most insensitive mutant followed by others (V219I, F255Y, WT, A251V and S264A). Further, in the presence of high concentrations of DCMU, identical transients were observed for all the mutants and the WT.The quantum yield of photochemistry of PS II, calculated from 1-(Fo/Fm), is in the range of 0.73 to 0.82 for the WT as well as for the mutants examined. Thus, in contrast to differences in the kinetics of the electron acceptor side of PS II, there were no significant differences in the maximum quantum yield of PS II, among the mutants tested. We suggest that earlier photochemistry yield values were much lower (0.4–0.6) than those reported here due to either higher measured values of Fo by instruments using camera shutters, or due to the use of cells grown in less than-optimal conditions.

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Study of photosystem 2 heterogeneity in the sulfur-deficient green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

A set of chlorophyll fluorescence methods, including PEA (Plant Efficiency Analyser), PAM (Pulse Amplitude Modulated fluorometer), and picosecond fluorometer, was employed to study PS 2 heterogeneity in sulfur deprived green algae Chlamydomonas reinhardtii. The regression method and JIP test were applied to analyze chlorophyll fluorescence kinetics. The fractions of PS 2 characterized by the energetic disconnection, smaller antenna size, elevated constant rate of primary photochemistry, and inability to maintain ΔpH-dependent energy dissipation increased essentially already after 12 h of incubation in sulfur depleted medium. The amount of PS 2 centers with reduced Q_A (closed state), Q_B-non-reducing centers with impaired water splitting function, and centers coupled to the plastoquinone pool with the slow cycle rate increased dramatically after 24 h period of deprivation. The mechanisms of PS 2 inactivation under sulfur deprivation are discussed.     

Studies of excitation energy transfer within the green alga Chlamydomonas reinhardtii and its mutants at 77 K

The 77 K picosecond fluorescence of intact Chlamydomonas reinhardtii exhibits a 680-nm band (F680) that can be identified with light-harvesting chlorophyll. Analysis of the time and spectral dependence of F680 reveal a forward transfer rate of 1/(15 ps) from this 680-nm species to photosystem II. The possibility of transfer through LHC I, the light-harvesting complex closely associated with photosystem I with a transfer time of 60 to 100 ps, is indicated by analysis of similar data in the 700–720 nm region. Simple kinetic models that account for the time dependence of the emissions F707, F703 and F715 are proposed.Based in part on a thesis submitted in partial fulfillment of the requirements for the Ph.D. Degree, University of Rochester (SL).     

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.