Analysis of the Chloroplast Protein Kinase Stt7 during State Transitions

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b₆f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light–induced reduction of this bond may occur through a transthylakoid thiol–reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b₆f assembly and heme biogenesis.     

nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii

Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.     

Carbon Supply and Photoacclimation Cross Talk in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO₂ availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO₂ maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO₂ reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii. This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability.Light sustains virtually all life on Earth through the process of photosynthesis. However, light can be very harmful for oxygenic photosynthetic organisms, as excess absorption can lead to the production of reactive oxygen species. In order to survive and grow, these organisms have developed various photoacclimation mechanisms operating on different time scales that protect the cell from photodamage. In the green alga Chlamydomonas reinhardtii, these mechanisms vary from negative phototaxis and multicomponent nonphotochemical quenching (NPQ) to a number of physiological and biochemical changes (Erickson et al., 2015). C. reinhardtii cells are around 10 μm in diameter, and a large part of their total volume is occupied by a single horseshoe-shaped chloroplast (Sager and Palade, 1957). The photosynthetic machinery responsible for the light reactions is located in thylakoid membranes and contains four major components: PSII, cytochrome b₆f, PSI, and ATP synthase. Both photosystems bind chlorophyll (Chl) and carotenoid (Car) and are composed of a core and several outer antennae pigment-protein complexes, the main function of which is light harvesting and its conversion into chemical energy. The PSII core is composed of D1, D2, CP43, and CP47 pigment-protein complexes and several smaller subunits, the number of which varies between organisms (Shi et al., 2012). The outer antenna contains the light-harvesting complex II (LHCII), which in C. reinhardtii is encoded by nine LHCBM genes, and the minor antennae CP26 and CP29 (Nield et al., 2000; Teramoto et al., 2001; Natali and Croce, 2015). These complexes are assembled together to form PSII-LHCII supercomplexes (Tokutsu et al., 2012; Drop et al., 2014). The PSI core is composed of a PSAA-PSAB heterodimer and a number of smaller subunits (Jensen et al., 2007), and in C. reinhardtii the LHCI antenna consists of nine LHCA proteins (Mozzo et al., 2010) that are associated with the core to form the PSI-LHCI complex (Stauber et al., 2009; Drop et al., 2011).The composition and organization of the thylakoid membrane is light dependent. The gene expression of different LHCs has been reported to be affected by light acclimation (Teramoto et al., 2002; Durnford et al., 2003; Yamano et al., 2008) and to be NAB1 regulated (Mussgnug et al., 2005). It has been observed that long-term high-light exposure of C. reinhardtii cells leads to a 50% decrease of Chl content (Neale and Melis, 1986; Bonente et al., 2012) and to changes in Chl-to-Car ratio (Niyogi et al., 1997a; Baroli et al., 2003; Bonente et al., 2012), suggesting reduction of the antenna size (Neale and Melis, 1986), although, in a more recent report (Bonente et al., 2012), it was concluded that the antenna size is not modulated by light in this alga. Recently, a dependence of the antenna components on the carbon availability also was reported. It was shown that, when cells grown in acetate are shifted from high to low CO₂ concentration, the functional antenna size of PSII decreases and a down-regulation of LHCBM6/8 occurs (Berger et al., 2014).In the short term, the main response to high light is the dissipation of energy absorbed in excess heat in a process called qE, or energy-dependent quenching, which is the fastest component of NPQ. In land plants, the main player in this process is the protein PsbS (Li et al., 2002, 2004), while in C. reinhardtii, the process is centered around LHCSR1 and LHCSR3 (Peers et al., 2009; Dinc et al., 2016). LHCSR3, the most studied of the two, is a pigment-protein complex that is expressed within 1 h of high-light exposure (Allorent et al., 2013) in combination with CO₂ limitation (Yamano et al., 2008; Maruyama et al., 2014). The qE onset is triggered by lumen acidification sensed by LHCSR3/1 (Bonente et al., 2011; Liguori et al., 2013; Tokutsu and Minagawa, 2013; Dinc et al., 2016).Cars are well known to be involved in photoprotection. They quench triplet Chl and scavenge singlet oxygen (¹O₂; Frank and Cogdell, 1996). In C. reinhardtii, the antioxidant role of xanthophylls is well illustrated by the mutant npq1 lor1 lacking lutein and zeaxanthin (Niyogi et al., 1997b). This mutant is deficient in qE, but compared with other qE-deficient mutants like npq4 (Peers et al., 2009) and npq5 (Elrad et al., 2002), which are LHCSR3 and LHCBM1 knockouts, respectively, it is extremely light sensitive, due to the absence of quenching of triplet Chl and ¹O₂ by zeaxanthin and lutein.Aquatic oxygenic photosynthetic organisms meet several challenges in CO₂ fixation (Moroney and Ynalvez, 2007). First, the diffusion of CO₂ in water is 10,000 times slower than in air. Second, the CO₂-fixing enzyme Rubisco is not selective for CO₂ and also binds oxygen, resulting in the process of photorespiration. Third, the form of inorganic carbon depends on the pH (i.e. in alkaline pH, it is HCO₃⁻, while in acidic pH, it is CO₂; Beardall, 1981; Gehl et al., 1987). This diminishes even further the availability of CO₂ in the cell. In order to overcome these CO₂ fixation barriers, algae have developed carbon-concentrating mechanisms (CCMs; Moroney and Ynalvez, 2007). The essence of these processes lies in the active pumping of inorganic carbon in the cell via a number of transporters that concentrate it in the pyrenoid, a ball-like structure containing Rubisco, Rubisco activase, and intrapyrenoid thylakoids and surrounded by a starch sheath. In the pyrenoid, HCO₃⁻ is converted to CO₂ by CARBONIC ANHYDRASE3 (CAH3; Blanco-Rivero et al., 2012; Sinetova et al., 2012) and then fixed by Rubisco in the Calvin-Benson-Bassham cycle. CAH3 also is suggested to provide HCO₃⁻ in the proximity of the oxygen-evolving complex, where it may function as a proton carrier, removing H⁺ from water splitting to avoid photoinhibition (Villarejo et al., 2002; Shutova et al., 2008).C. reinhardtii also can grow mixotrophically using alternative organic carbon sources present in its environment. For example, it can take up acetate, which is then incorporated into the citric cycle, producing reducing equivalents and CO₂ (Johnson and Alric, 2012), and into the glyoxylate cycle, producing malate (Lauersen et al., 2016). In the presence of acetate, it has been reported that CO₂ uptake and oxygen evolution were decreased by half under saturating CO₂ and light intensities without affecting PSII efficiency, respiration, and cell growth (Heifetz et al., 2000). In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions (Chapman et al., 2015). Furthermore, acetate can replace PSII-associated HCO₃⁻, reducing ¹O₂ formation and, therefore, acting as a photoprotector during high-light acclimation (Roach et al., 2013).In short, high-light acclimation is a complex, multicomponent process that happens on different time scales. Furthermore, it is embedded in the overall metabolic network and is potentially influenced by different nutrients and metabolic states. A thorough understanding of this process and its regulation is crucial for fundamental research and applications. To determine if different carbon supply conditions trigger different light acclimation strategies and photoprotective responses, we systematically studied C. reinhardtii cells grown in mixotrophic, photoautotrophic, and high-CO₂ photoautotrophic conditions in different light intensities.We show that C. reinhardtii cells use different strategies to acclimate to high light depending on the carbon availability and trophic status. These results underline the strong connection between metabolism and light acclimation responses and reconcile the data from various reports. Furthermore, our study demonstrates how, in a dynamic system such as C. reinhardtii, a single change in growth conditions has large effects at multiple levels.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii

We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.     

Genome-Wide Characterization of Genetic Variation in the Unicellular, Green Alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a model system for studying cilia, photosynthesis, and other core features of eukaryotes, and is also an emerging source of biofuels. Despite its importance to basic and applied biological research, the level and pattern of genetic variation in this haploid green alga has yet to be characterized on a genome-wide scale. To improve understanding of C. reinhardtii's genetic variability, we generated low coverage whole genome resequencing data for nearly all of the available isolates of this species, which were sampled from a number of sites in North America over the past ～70 years. Based on the analysis of more than 62,000 single nucleotide polymorphisms, we identified two groups of isolates that represent geographical subpopulations of the species. We also found that measurements of genetic diversity were highly variable throughout the genome, in part due to technical factors. We studied the level and pattern of linkage disequilibrium (LD), and observed one chromosome that exhibits elevated LD. Furthermore, we detected widespread evidence of recombination across the genome, which implies that outcrossing occurs in natural populations of this species. In summary, our study provides multiple insights into the sequence diversity of C. reinhardtii that will be useful to future studies of natural genetic variation in this organism.     

Acclimation of Photosynthetic Light Reactions during Induction of Inorganic Carbon Accumulation in the Green Alga Chlamydomonas reinhardtii

Cells of the unicellular green algae Chlamydomonas reinhardtii were grown in high dissolved inorganic carbon (DIC) concentrations (supplied with 50 milliliters per liter CO₂[g]) and transferred to low DIC concentrations (supplied with ≤ 100 microliters per liter CO₂[g]). Immediately after transfer from high to low DIC the emission of photosystem II related chlorophyll a fluorescence was substantially quenched. It is hypothesized that the suddenly induced inorganic carbon limitation of photosynthesis resulted in a phosphorylation of LHCII, leading to the subsequent state 1 to state 2 transition. After 2 hours of low-DIC acclimation, 77 K fluorescence measurements revealed an increase in the fluorescence emitted from photosystem I, due to direct excitation, suggesting a change in photosystem II/photosystem I stoichiometry or an increased light harvesting capacity of photosystem I. After 5 to 6 hours of acclimation a considerable increase in spillover from photosystem II to photosystem I was observed. These adjustments of the photosynthetic light reactions reached steady-state after about 12 hours of low DIC treatment. The quencher of fluorescence could be removed by 5 minutes of dark treatment followed by 5 minutes of weak light treatment, of any of four different light qualities. It is hypothesized that this restoration of fluorescence was due to a state 2 to state 1 transition in low-DIC acclimated cells. A decreased ratio of violaxanthin to zeaxanthin was also observed in 12 hour low DIC treated cells, compared with high DIC grown cells. This ratio was not coupled to the level of fluorescence quenching. The role of different processes during the induction of a DIC accumulating mechanism is discussed.     

Alteration of the Cell Surface during the Vegetative Cell Cycle of the Unicellular Green Alga Chlamydomonas reinhardtii

Alterations of the cell surface during the vegetative cell cycleof the unicellular green alga Chlamydomonas reinhardtii wereinvestigated using polyclonal antibodies against the purifiedand subsequently deglycosylated insoluble cell wall componentand against a 100 kDa polypeptide of the deglycosylated, chaotrope-solublewall fraction, respectively. Both antibodies recognized epitopeswithin the non-glycosylated domains of a ‘150 kDa’chaotrope-soluble glycoprotein (=GP3B) localized in the outerlayers of the C. reinhardtii cell wall. Immunofluorescence studiesindicated that both antibodies reacted with the surface of ‘late’sporangia (harvested 1 h before liberation of the zoospores),but not with the cell surfaces of released zoospores, growingcells and young sporangia, respectively. After pretreatmentwith aqueous LiCl, however, the cell surfaces of zoospores,growing cells and young sporangia became accessible to theseparticular antibodies. Highly purified preparations of the insolublewall fraction revealed strong immunofluorescence with both antibodiesbut not with the corresponding preimmune sera. Based on thesedata, we concluded that the antigenic sites of the insolubleglycoprotein framework of the C. reinhardtii wall are maskedby LiCl-soluble glycoproteins in single cell stages and youngsporangia, but not or to a lesser extent in the case of themother walls of ‘late’ sporangia. The conclusionwas supported by findings that (I) the multilayered structureof the mother-cell wall was disturbed in ‘late’,but not in young sporangia and that (II) the amounts of chaotropesolublecell wall glycoproteins present in the LiCl-extracts from intactsporangia decreased during ripening of the sporangia. (Received January 10, 1996; Accepted May 27, 1996)     

Relief of Arsenate Toxicity by Cd-Stimulated Phytochelatin Synthesis in the Green Alga Chlamydomonas reinhardtii

In most photosynthetic organisms, inorganic arsenic taken up into the cells inhibits photosynthesis and cellular growth. In a green alga, Chlamydomonas reinhardtii, 0.5 mM arsenate inhibited photosynthesis almost completely within 30 min. However, in cells acclimated with a sublethal concentration (0.05 to 0.1 mM) of Cd, the inhibition of photosynthesis at 30 min after the addition of arsenate was relieved by more than 50%. The concentrations of arsenic incorporated into the cells were not significantly different between the Cd-acclimated and the non-acclimated cells. The Cd-acclimated cells accumulated Cd and synthesized phytochelatin (PC) peptides, which are known to play an important role in detoxification of heavy metals in plants. By the addition of an inhibitor of glutathione (an intermediate in the PC biosynthetic pathway) biosynthesis, buthionine sulfoximine, cells lost not only Cd tolerance but also arsenate tolerance. These results suggest that glutathione and/or PCs synthesized in Cd-acclimated cells are involved in mechanisms of arsenate tolerance. The authors contributed equally to this work.     

The Cytotoxic Effects of Camptothecin and Mastoparan on the Unicellular Green Alga Chlamydomonas reinhardtii

We have recently reported that protease inhibitors affecting the activity of the proteasome cause necrotic cell death in Chlamydomonas reinhardtii instead of inducing apoptosis as shown for some mammalian cell lines. Therefore, we have studied other well‐known inducers of apoptosis in mammalian cells for their effects on C. reinhardtii cells. Mastoparan caused rapid cell death without a prominent lag‐phase under all growth conditions, whereas the cytotoxic effect of the topoisomerase I inhibitor camptothecin exclusively occurred during the cell‐division phase. Essentially no differences between wall‐deficient and wild‐type cells were observed with respect to dose‐response and time‐course of camptothecin and mastoparan. In cultures of the wall‐deficient strain, cell death was accompanied by swelling and subsequent disruption of the cells, established markers of necrosis. In case of the wild‐type strain, camptothecin and mastoparan caused accumulation of apparently intact, but dead cells instead of cell debris due to the presence of the wall. Both in cultures of the wall‐deficient and the wild‐type strains, cell death was accompanied by an increase of the protein concentration in the culture medium indicating a lytic process like necrosis. Taking together, we have severe doubts on the existence of an apoptotic program in case of C. reinhardtii.     

Blue Light-Induced Lethality of a Cell Wall-Deficient Mutant of the Unicellular Green Alga Chlamydomonas reinhardtii

When synchronized cultures of a cell wall-deficient Chlamydomonasreinhardtii mutant strain were grown under heterotrophic conditionsand subsequently transferred to the light, a considerable decreaseof the cell number was observed during transition to the celldivision phase. Lethality of the wall-deficient cells was inducedby blue light, but not by red or far-red light, and could notbe prevented by addition of the photosystem II inhibitor DCMU.The light-induced lethality was found to be restricted to wall-deficientcells which were agitated by bubbling with filtered air or nitrogenor vigorously shaken during the transition to the cell divisionphase. Therefore, a (blue) light-induced sensitivity to anymechanical stress seems to be the cause for cell death. In heterotrophicallygrowing cultures of the Chlamydomonas wild-type, illuminationwith blue or white light did not cause a decrease of the cellnumber but only a delay of cell divisions. The latter effectwas also observed in case of the wall-deficient mutant. Bothblue light effects are observed during the transition to thecell division phase and can be induced during the same periodof the cell cycle. Furthermore, the (blue) light-induced lethalityof wall-deficient cells was found to be prevented when the transitionto the cell division phase was inhibited by addition of antibiotics.Therefore, we assume that there is a connection between theblue light-induced sensitivity to mechanical stress and theblue light-induced delay of cell divisions. (Received September 3, 1993; Accepted November 12, 1993)     

Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii

The “Phot” protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 μs with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.     

Functional Rearrangement of the Light-Harvesting Antenna upon State Transitions in a Green Alga

State transitions in the green alga Chlamydomonas reinhardtii serve to balance excitation energy transfer to photosystem I (PSI) and to photosystem II (PSII) and possibly play a role as a photoprotective mechanism. Thus, light-harvesting complex II (LHCII) can switch between the photosystems consequently transferring more excitation energy to PSII (state 1) or to PSI (state 2) or can end up in LHCII-only domains. In this study, low-temperature (77 K) steady-state and time-resolved fluorescence measured on intact cells of Chlamydomonas reinhardtii shows that independently of the state excitation energy transfer from LHCII to PSI or to PSII occurs on two main timescales of <15 ps and ∼100 ps. Moreover, in state 1 almost all LHCIIs are functionally connected to PSII, whereas the transition from state 1 to a state 2 chemically locked by 0.1 M sodium fluoride leads to an almost complete functional release of LHCIIs from PSII. About 2/3 of the released LHCIIs transfer energy to PSI and ∼1/3 of the released LHCIIs form a component designated X-685 peaking at 685 nm that decays with time constants of 0.28 and 5.8 ns and does not transfer energy to PSI or to PSII. A less complete state 2 was obtained in cells incubated under anaerobic conditions without chemical locking. In this state about half of all LHCIIs remained functionally connected to PSII, whereas the remaining half became functionally connected to PSI or formed X-685 in similar amounts as with chemical locking. We demonstrate that X-685 originates from LHCII domains not connected to a photosystem and that its presence introduces a change in the interpretation of 77 K steady-state fluorescence emission measured upon state transitions in Chalamydomonas reinhardtii.     

UV-A Inhibition of Alternative Respiration in Pea Leaves and a Unicellular Green Alga Chlamydomonas reinhardtii

Total respiration (v_T) increased after exposure to UV, but a decrease in the capacity of SHAM-sensitive-alternative respiration (V_alt) was accompanied by an increase in residual respiration (v_res). The capacity for CN sensitive-cytochrome c respiration (V_cyt) was not inhibited by UV-A. After 4 h of irradiation of high-CO₂-grown cells of Chlamydomonas reinhardtii with UV-A (2 μW. CM^?2) in the presence of white light (300μE.m^?2.s^?1), the capacity of Vast was reduced from 10 to 4 μmol O₂. mg^?1Chl.h^?1, a 60 % reduction. After a similar exposure to UV-A, the capacity of V_alt in pea leaves was reduced from 13 to 5 μmol O₂.g^?1 fr wt.h^?1. Exposure to UV-C was not inhibitory, but UV-B caused up to 25% inhibition of the V^alt. Twenty to 48 h after exposure to UV-A radiation, the capacity of alternative respiration had recovered. UV-A inhibition of the alternative respiration was consistent with UV-A absorption by quinones, except that UV-A did not inhibit the cyt c pathway of electron transport that also involves the ubiquinones.     

Phosphorylation of axonemal proteins in Chlamydomonas reinhardtii.

In order to determine whether microtubular proteins of flagellar axonemes were phosphorylated, cells of Chlamydomonas reinhardtii were grown in medium containing [32P]orthophosphate for several generations. Only one (alpha subunit) of the two tubulin polypeptides separated by Na dodecyl-SO4-polyacrylamide gel electrophoresis appeared labeled, as detected by autoradiography of the dried gel. 3H- and 32P-labeled alpha tubulin subunit purified by preparative Na dodecyl-SO4-polyacrylamide gel electrophoresis and Na dodecyl-SO4-hydroxyapatite chromatography contained about 0.2 mol of phosphate per mol of polypeptide. Upon partial acid hydrolysis, radioactivity could be accounted for as serine and threonine phosphate. By altering the conditions of the Na dodecyl-SO4-polyacrylamide gel electrophoresis is was possible to resolve the purified alpha-tubulin subunit into five or more components: a major band comprising approximately 65% of the total mass, not phosphorylated, and four or more minor bands comprising together 35% of the mass. Among the minor components at least two were phosphorylated.     

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12–15 core and 4–9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.