CHANGE OF UBIQUITINATED PROTEINS DURING THE CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYTA)1

Changes in the amount of heat shock-related ubiquitinated proteins in Chlamydomonas were investigated during the cell cycle and gamete induction. In a division-synchronized culture induced by periodic illumination, the amount of the 28-kDa ubiquitinated protein increased during the dark phase. This increase correlated with the increase of total DNA. Such an increase was repressed when nuclear DNA replication was inhibited with aphidicolin. These results suggest that ubiquitination to form the 28-kDa protein is involved in nuclear DNA replication or during the cell cycle. The amount of 31-kDa ubiquitinated protein gradually increased throughout the light phase and decreased in the dark phase. The amount of 28-kDa ubiquitinated protein also increased during gamete induction caused by nitrogen starvation, while that of the 31-kDa did not. These results suggest that the change of ubiquitination of 28-kDa protein mat play a fundamental role in the cell cycle and gamete induction in Chlamydomonas.     

Cross-reconstitution of the extrinsic proteins and Photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea

Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.     

The thioredoxin superfamily in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The thioredoxin (TRX) superfamily includes redox proteins such as thioredoxins, glutaredoxins (GRXs) and protein disulfide isomerases (PDI). These proteins share a common structural motif named the thioredoxin fold. They are involved in disulfide oxido-reduction and/or isomerization. The sequencing of the Arabidopsisgenome revealed an unsuspected multiplicity of TRX and GRX genes compared to other organisms. The availability of full Chlamydomonasgenome sequence offers the opportunity to determine whether this multiplicity is specific to higher plant species or common to all photosynthetic eukaryotes. We have previously shown that the multiplicity is more limited in Chlamydomonas for TRX and GRX families. We extend here our analysis to the PDI family. This paper presents a comparative analysis of the TRX, GRX and PDI families present in Arabidopsis,Chlamydomonas and Synechocystis. The putative subcellular localization of each protein and its relative expression level, based on EST data, have been investigated. This analysis provides a large overview of the redox regulatory systems present in Chlamydomonas. The data are discussed in view of recent results suggesting a complex cross-talk between the TRX, GRX and PDI redox regulatory networks.     

Transmembrane signaling in cilia and flagella

Summary Ciliary and flagellar membranes are dynamic. Ciliary and flagellar membranes have diverged widely during evolution and perform many specialized functions. Transmembrane signaling is an important component of the function of ciliary and flagellar surfaces in general. In this review, I discuss some of the functions performed by ciliary and flagellar surfaces and I present three different ciliary and flagellar signaling systems associated with rather different dynamic events performed by ciliary and flagellar surfaces. Two of these are associated withChlamydomonas flagella and one is associated with vertebrate olfactory cilia. Calcium regulation of protein phosphorylation appears to be important in regulating glycoprotein movements in theChlamydomonas flagellar membrane. Changes in levels of cAMP and cAMP-dependent protein phosphorylation are clearly central to the signaling associated with mating events in gametic flagella ofChlamydomonas, although calcium clearly has an important, if poorly understood, role to play. There is no known role for G proteins in flagellar membrane events inChlamydomonas. In contrast, mammalian olfactory cilia possess an odorant activated, G protein regulated adenylate cyclase and conductance channels that are directly gated by cyclic nucleotides. A second class of odorants that do not affect adenylate cyclase activity appear to act through G protein activated phospholipase C and changes in IP₃ second messenger levels. These examples demonstrate the diversity in the signaling pathways associated with ciliary and flagellar membranes.Abbreviations CaPK-2 calcium-dependent protein kinase - db-cAMP dibutyryl cAMP - Fab fragment antigen binding - IgE immunoglobulin E - IP₃ myo-inositol trisphosphate - IP₄ myo-inositol tetrakisphosphate - OBP odorant binding protein - PIP₂ phosphoinositol bisphosphate - TFP trifluoperazine - WGA wheat germ agglutinin     

Cellular aggregation in Chlamydomonas (Chlorophyceae) is chimaeric and depends on traits like cell size and motility

How the variation in phenotypic traits like cell size and motility impacts predator-induced cellular aggregation is not known. Furthermore the genetic composition of cell groups in mixed populations of Chlamydomonas has not been investigated. An examination of these two questions will not only enhance our understanding of Chlamydomonas ecology, but also shed light on the primordial steps before integrated multicellular groups were established. Group living comes with viability and reproductive costs and it is not known how these are shared if groups are genetically heterogeneous. We observed that the natural predator Peranema trichophorum (Euglenoidea) induced clumping in Chlamydomonas. When co-cultured with P. trichophorum cells protected themselves by forming facultative groups (reverting back to a unicellular lifestyle once predators were removed). The dynamics of group formation in different Chlamydomonas species and strains correlated with cell size and swimming speed. Small or less motile strains aggregated more readily than large, fast-swimming ones. Interestingly, Chlamydomonas groups were both intra-species and inter-species chimaeric. This suggests that the predator-induced group formation in Chlamydomonas involved cells coming together rather than staying together and during aggregation cells showed little or no discrimination between self and non-self. These data demonstrate that the dynamics of cell aggregation, in unicellular volvocines at least, depends on phenotypic traits like cell size and motility and high genetic relatedness is not mandatory at this initial stage. These findings further our understanding of aggregation in mixed Chlamydomonas populations and have implications for understanding the very first steps on the road to simple multicellularity.     

A marine Chlamydomonas sp. emerging as an algal model

The freshwater microalga Chlamydomonas reinhardtii, which lives in wet soil, has served for decades as a model for numerous biological processes, and many tools have been introduced for this organism. Here, we have established a stable nuclear transformation for its marine counterpart, Chlamydomonas sp. SAG25.89, by fusing specific cis‐acting elements from its Actin gene with the gene providing hygromycin resistance and using an elaborated electroporation protocol. Like C. reinhardtii, Chlamydomonas sp. has a high GC content, allowing reporter genes and selection markers to be applicable in both organisms. Chlamydomonas sp. grows purely photoautotrophically and requires ammonia as a nitrogen source because its nuclear genome lacks some of the genes required for nitrogen metabolism. Interestingly, it can grow well under both low and very high salinities (up to 50 g · L^‐1) rendering it as a model for osmotolerance. We further show that Chlamydomonas sp. grows well from 15 to 28°C, but halts its growth at 32°C. The genome of Chlamydomonas sp. contains some gene homologs the expression of which is regulated according to the ambient temperatures and/or confer thermal acclimation in C. reinhardtii. Thus, knowledge of temperature acclimation can now be compared to the marine species. Furthermore, Chlamydomonas sp. can serve as a model for studying marine microbial interactions and for comparing mechanisms in freshwater and marine environments. Chlamydomonas sp. was previously shown to be immobilized rapidly by a cyclic lipopeptide secreted from the antagonistic bacterium Pseudomonas protegens PF‐5, which deflagellates C. reinhardtii.     

Application of quantitative immunoprecipitation combined with knockdown and cross‐linking to Chlamydomonas reveals the presence of vesicle‐inducing protein in plastids 1 in a common complex with chloroplast HSP90C

Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein–protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein–protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle‐inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross‐linking step (‐X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross‐linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions.     

Extremely low polymerizability of a highly-divergent Chlamydomonas actin (NAP)

Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.     

N‐ACYL HOMOSERINE LACTONe LACTONASE,AiiA, INACTIVATION OF QUORUM‐SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE1

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N‐acyl homoserine lactone (AHL) bacterial quorum‐sensing (QS) signals and alter QS‐regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal‐mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR‐stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal‐mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL‐producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.     

Characterization of a Chlamydomonas reinhardtii gene encoding a protein of the DNA photolyase/blue light photoreceptor family

The organization and nucleotide sequence of a gene from Chlamydomonas reinhardtii encoding a member of the DNA photolyase/blue light photoreceptor protein family is reported. A region of over 7 kb encompassing the gene was sequenced. Northern analysis detected a single 4.2 kb mRNA. The gene consists of eight exons and seven introns, and encodes a predicted protein of 867 amino acids. The first 500 amino acids exhibit significant homology with previously sequenced DNA photolyases, showing the closest relationship to mustard (Sinapis alba) photolyase (43% identity). An even higher identity, 49%, is obtained when the Chlamydomonas gene product is compared to the putative blue-light photoreceptor (HY4) from Arabidopsis thaliana. Both the Chlamydomonas and the Arabidopsis proteins differ from the well characterized DNA photolyases in that they contain a carboxyl terminal extension of 367 and 181 amino acids, respectively. However, there is very little homology between the carboxyl terminal domains of the two proteins. A previously isolated Chlamydomonas mutant, phrl, which is deficient in DNA photolyase activity, especially in the nucleus, was shown by RFLP analysis not to be linked to the gene we have isolated. We propose this gene encodes a candidate Chlamydomonas blue light photoreceptor.     

Calmodulin Sensitivity of the Flagellar Membrane Adenylate Cyclase and Signaling of Motile Responses by cAMP in Gametes of Chlamydomonas reinhardtii

Cyclic AMP (cAMP) has been shown to be a primary signal of the agglutination-induced mating events of flagellar tip activation, cell wall loss, and mating structure activation in the unicellular alga Chlamydomonas reinhardtii (Pasquale and Goodenough, Cell Biol. 105 (1987), 2279–2293). The flagellar membrane adenylate cyclase of Chlamydomonas is here shown to be inhibited in vitro by EGTA, La³⁺, and trifluoperazine, and to be stimulated in the presence of calcium by incubation with exogenous calmodulin. Also, the motility of detergent-extracted models of Chlamydomonas is shown to be enhanced by cAMP. These observations suggest the hypothesis that the twitching motility characteristic of agglutinating Chlamydomonas gametes may be signaled by cAMP produced locally within the flagella by a calmodulin-sensitive adenylate cyclase.     

The Lost Neuromotor Apparatus of Chlamydomonas: Rediscovered1,2

ABSTRACT. Early light microscopic studies of the biflagellate green alga Chlamydomonas revealed a fibrous system, the neuromotor apparatus, which appeared to link physically the flagellar apparatus to the cell nucleus. Following the development of the electron microscope, the existence of a neuromotor apparatus in Chlamydomonas was cast into doubt since it was not observed in studies carried out at ultrastructural resolution. Here we show, by indirect immunofluorescence, using monoclonal antibodies and electorn microscopy employing refined specimen preparation and staining techniques, that the neuromotor apparatus of Chlamydomonas does indeed exist. The functional significance of this system is discussed in light of both historic proposals and recent experimental findings.     

COPPER SORPTION AND RELEASE BY CYCLOTELLA MENEGHINIANA (BACILLARIOPHYCEAE) AND CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Initial Cu⁺⁺ sorption by Cyclotella meneghiniana Kütz. (Cu⁺⁺-sensitive) and Chlamydomonas reinhardtii Dangeard (Cu⁺⁺-resistant) was rapid in the first 5 min of Cu⁺⁺ incubation with little sorption after 2 h. On a cell to cell basis, Cyclotella sorbed ca. five times more Cu⁺⁺ from the medium than Chlamydomonas. In MBL medium with EDTA Cyclotella and Chlamydomonas cells sorbed 21.0 and 4.41 nM Cu⁺⁺/10⁶ cells respectively in 6 h with 0.3 mg Cu⁺⁺/l in the medium. Proportionally similar quantities of Cu⁺⁺ were sorbed when the cells were Cu⁺⁺ incubated in MBL + citrate or filtered lake water. Cleaned cell walls of Cyclotella sorbed little Cu⁺⁺ (1.7 nM/10⁶ cells) as compared to living cells (17.5 nM Cu⁺⁺/10⁶ cells) in 3 h. Therefore, in living Cyclotella most of the Cu⁺⁺ taken up must be absorbed by the protoplasm or perhaps by the organic layer surrounding the silica wall. Cleaned cell walls of Chlamydomonas sorbed 3.5 nM Cu⁺⁺/10⁶ cells and living Chlamydomonas cells sorbed 2.6 nM Cu⁺⁺/10⁶ cells. This indicates that most of the Cu⁺⁺ sorbed by Chlamydomonas cells remained bound to the cell wall and probably did not readily enter into the protoplasm: When placed in Cu⁺⁺ free medium after Cu⁺⁺ incubation, Cyclotella and Chlamydomonas cells released 46 and 59% respectively of the Cu⁺⁺ sorbed.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Chloroplasts of the green alga Chlamydomonas reinhardtii possess at least four distinct stromal processing proteases

The majority of the proteins in the chloroplast are encoded in the nucleus and synthesised in the cytoplasm as precursors with N-terminal extensions. These targeting sequences guide the precursor proteins into the chloroplast where they are immediately cleaved off by a stromal processing protease (SPP). It is commonly assumed that in higher plant chloroplasts one general SPP processes almost all imported precursor proteins. In the green alga Chlamydomonas, however, there exist several different SPPs which process the various Chlamydomonas precursor proteins. The seven precursor proteins investigated here, which were all correctly imported into isolated chloroplasts, could be divided into two groups: Four precursor proteins were cleaved correctly when processed in vitro with an extract of stromal proteins. Four different SPPs were found in Chlamydomonas chloroplasts to be responsible for the processing of this class of precursors and these four activities were separated chromatographically, characterised and further distinguished by their sensitivity to different inhibitors. The three precursors of the second group were degraded completely by unidentified enzyme(s) present in the stromal extract. Degradation of these precursors was dependent on their conformational integrity as well as on the redox state in the stroma. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cabI/II genes that is regulated during the cell cycle and in response to illumination

In the green unicellular alga Chlamydomonas reinhardtii, as in higher plants, the expression of the genes encoding the chlorophyll a/b-binding (CAB) polypeptides associated with photosystem I (PSI) and photosystem II (PSII) is regulated by endogenous (circadian clock) and exogenous signals (light and temperature). The circadian clock ensures that the oscillation in the levels of the different cab mRNAs is continuously kept in phase with light/dark (LD) cycles and is maximal by the middle of the day. On the other hand, light controls the amplitude of the oscillations. We report here the cloning and characterization of the C. reinhardtii LI818 gene, which identifies a CAB-related polypeptide and whose expression is regulated quite differently from the cabI/II genes. We show: (1) that in LD synchronized Chlamydomonas cells LI818 mRNA accumulation is subject to dual regulation that involves separable regulation by light and an endogenous oscillator; (2) that LI818 mRNA is fully expressed several hours before the cab I/II mRNAs and that the latter accumulate concomitantly; (3) that blocking the electron flow through PSII using DCMU prevents cells from accumulating cab I/II mRNAs but not LI818 mRNA and (4) that the accumulation of LI818 mRNA is abolished by blocking cytoplasmic protein synthesis, suggesting that these regulatory mechanisms are mediated by labile proteins.     

DIEL RHYTHM OF ALGAL PHOSPHATE UPTAKE RATES IN P‐LIMITED CYCLOSTATS AND SIMULATION OF ITS EFFECT ON GROWTH AND COMPETITION1

Oscillations in the phosphate (P_i) uptake rates for three species of green algae were examined in a P‐limited cyclostat. For Ankistrodesmus convolutus Corda and Chlorella vulgaris Beyerinck, the P_i uptake rates increased during the daytime and decreased at night. In contrast, Chlamydomonas sp. exhibited the opposite uptake pattern. Cell densities also oscillated under a light:dark cycle, dividing at a species‐specific timing rather than continuously. In general, the cell densities exhibited an inverse relationship with the P_i uptake rates. A competition experiment between A. convolutus and C. vulgaris in a P‐limited cyclostat resulted in the dominance of C. vulgaris, regardless of the relative initial cell concentrations. Chlorella vulgaris also dominated in a mixed culture with Chlamydomonas sp., irrespective of the initial seeding ratio and dilution rate. However, Chlamydomonas sp. and A. convolutus coexisted in the competition experiment with gradual decrease of Chlamydomonas sp. when equally inoculated. Mathematical expressions of the oscillations in the P_i uptake rate and species‐specific cell division gate were used to develop a simulation model based on the Droop equation. The simulation results for each of the species conformed reasonably well to the experimental data. The results of the competition experiments also matched the competition simulation predictions quite well, although the experimental competition was generally more delayed than the simulations. In conclusion, the model simulation that incorporated the effect of diel rhythms in nutrient uptake clearly demonstrated that species diversity could be enhanced by different oscillation patterns in resource uptake, even under the condition of limitation by the same resource.     

Enhanced Tolerance Against Salt-Stress and Freezing-Stress of Escherichia coli Cells Expressing Algal bbc1 Gene

The expression of the eukaryotic bbc1 (breast basic conserved) gene (the bbc1 gene of the marine green alga Chlamydomonas sp. W-80 strain) enhanced the tolerance against salt-stress and freezing-stress in E. coli cells. The expression of the BBC1 protein in the E. coli cells carrying the algal bbc1 gene and that in the Chlamydomonas W-80 cells were examined by Western blotting analysis. The result suggests that the eukaryotic BBC1 protein expressed in the E. coli cells has a protective function against the cellular dehydration. Received: 24 April 2000 / Accepted: 21 August 2000