Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.     

Architecture and evolution of subtelomeres in the unicellular green alga Chlamydomonas reinhardtii

In most eukaryotes, subtelomeres are dynamic genomic regions populated by multi-copy sequences of different origins, which can promote segmental duplications and chromosomal rearrangements. However, their repetitive nature has complicated the efforts to sequence them, analyse their structure and infer how they evolved. Here, we use recent genome assemblies of Chlamydomonas reinhardtii based on long-read sequencing to comprehensively describe the subtelomere architecture of the 17 chromosomes of this model unicellular green alga. We identify three main repeated elements present at subtelomeres, which we call Sultan, Subtile and Suber, alongside three chromosome extremities with ribosomal DNA as the only identified component of their subtelomeres. The most common architecture, present in 27 out of 34 subtelomeres, is a heterochromatic array of Sultan elements adjacent to the telomere, followed by a transcribed Spacer sequence, a G-rich microsatellite and transposable elements. Sequence similarity analyses suggest that Sultan elements underwent segmental duplications within each subtelomere and rearranged between subtelomeres at a much lower frequency. Analysis of other green algae reveals species-specific repeated elements that are shared across subtelomeres, with an overall organization similar to C. reinhardtii. This work uncovers the complexity and evolution of subtelomere architecture in green algae.     

Characterization of the LI818 polypeptide from the green unicellular alga Chlamydomonas reinhardtii

The LI818 gene from Chlamydomonas encodes a polypeptide that is related to the chlorophyll a/b-binding proteins (CAB) of higher plants and green algae. However, despite this relatedness, LI818 gene expression is not coordinated with that of cab genes and is regulated differently by light, suggesting a different role for LI818 polypeptide. We show here that, in contrast to CAB polypeptides, LI818 polypeptide is not tightly embedded into the thylakoid membranes and is localized in stroma-exposed regions. Moreover, during chloroplast development, LI818 polypeptide accumulates before CAB polypeptides. We also show that the LI818 polypeptide forms with certain chlorophyll a/c-binding proteins (CAC) from the haptophyte Isochrysis galbana and the diatom Cyclotella cryptica a natural group that is distinct from those constituted by CAB, CAC and the chlorophyll a/a-binding proteins (CAA). Such an association suggests a very ancient origin for this group of polypeptides, which predates the division of the early photosynthetic eukaryotes into green (chlorophyte), red (rhodophyte) and brown (chromophyte) algae. Possible roles for the LI818 polypeptide are discussed.     

Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii

The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.     

Multiple facets of anoxic metabolism and hydrogen production in the unicellular green alga Chlamydomonas reinhardtii

Many microbes in the soil environment experience micro-oxic or anoxic conditions for much of the late afternoon and night, which inhibit or prevent respiratory metabolism. To sustain the production of energy and maintain vital cellular processes during the night, organisms have developed numerous pathways for fermentative metabolism. This review discusses fermentation pathways identified for the soil-dwelling model alga Chlamydomonas reinhardtii, its ability to produce molecular hydrogen under anoxic conditions through the activity of hydrogenases, and the molecular flexibility associated with fermentative metabolism that has only recently been revealed through the analysis of specific mutant strains.     

The transformation of the unicellular alga Chlamydomonas reinhardtii by electroporation

The cell wall-lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase gene hpt as the selective marker and the Tn5 transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10(6) mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10(3) HygR transformants per 10(6) recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation of Ch. reinhardtii with heterologous genes are discussed.     

Ultrastructural,cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

Summary— The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.     

Rapid biotransformation of arsenate into oxo-arsenosugars by a freshwater unicellular green alga, Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

Predicting the physiological role of circadian metabolic regulation in the green alga Chlamydomonas reinhardtii

Although the number of reconstructed metabolic networks is steadily growing, experimental data integration into these networks is still challenging. Based on elementary flux mode analysis, we combine sequence information with metabolic pathway analysis and include, as a novel aspect, circadian regulation. While minimizing the need of assumptions, we are able to predict changes in the metabolic state and can hypothesise on the physiological role of circadian control in nitrogen metabolism of the green alga Chlamydomonas reinhardtii.     

Subcellular distribution of 14-3-3 proteins in the unicellular green alga Chlamydomonas reinhardtii.

A polyclonal antibody was raised against a recombinant Chlamydomonas 14-3-3-beta-galactosidase (beta-Gal) fusion protein and characterized for its epitope specificity towards the corresponding Chlamydomonas 14-3-3 protein by scan-peptide analysis. This antibody recognized four Chlamydomonas polypeptides with apparent molecular masses 32, 30, 27, and 24 kDa, which also reacted with the antiserum depleted of anti-(Escherichia coli beta-Gal) IgG, but not with the corresponding preimmune serum or the antiserum preincubated with purified 14-3-3 proteins. Western-blot analyses performed with the antibody depleted of anti-(beta-Gal) IgG revealed that more or less pronounced levels of 14-3-3 proteins were present in all subcellular fractions of Chlamydomonas reinhardtii except the nuclei. The highest levels of 14-3-3 protein were observed in the cytosol and microsomal fraction. The 30-kDa isoform was predominant in the cytosol, whereas the 27-kDa isoform was prevalent in the microsomes. When microsomal membranes were separated by sucrose-density-gradient centrifugation, Western-blot analysis revealed distinct patterns of 14-3-3 isoforms in the endoplasmic reticulum, dictyosome, and plasma membrane fractions identified by marker enzyme activities. These findings indicate that the four 14-3-3 proteins of C. reinhardtii differentially interact with endoplasmic reticulum, dictyosomes, and plasma membrane.     

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus-associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post-embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A-gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.     

Structure and expression of the ornithine decarboxylase gene of Chlamydomonas reinhardtii

A cDNA was cloned encoding ornithine decarboxylase (ODC) of the unicellular green alga Chlamydomonas reinhardtii. The polypeptide consists of 396 amino acid residues with 35–37% sequence identity to other eukaryotic ODCs. As indicated by the phylogenetic tree calculated by neighbour joining analysis, the Chlamydomonas ODC has the same evolutionary distances to the ODCs of higher plants and mammalians. The Chlamydomonas ODC gene contains three introns of 222, 133, and 129 bp, respectively. As revealed by Northern-blot analyses, expression of the Chlamydomonas ODC gene is neither altered throughout the vegetative cell cycle nor modulated by exogenous polyamines.     

Proteomic analysis of high-CO(2)-inducible extracellular proteins in the unicellular green alga, Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a wide range of CO(2) concentrations through the regulation of a CO(2)-concentrating mechanism (CCM). By proteomic analysis, here we identified the proteins which were specifically accumulated under high-CO(2) conditions in a cell wall-less strain of C. reinhardtii which release their extracellular matrix into the medium. When the CO(2) concentration was elevated from the ambient air level to 3% during culture, the algal growth rate increased 1.5-fold and the composition of extracellular proteins, but not intracellular soluble and insoluble proteins, clearly changed. Proteomic analysis data showed that the levels of 22 of 129 extracellular proteins increased for 1 and 3 d and such multiple high-CO(2)-inducible proteins include gametogenesis-related proteins and hydroxyproline-rich glycoproteins. However, we could not prove the induction of gametogenesis under high-CO(2) conditions, suggesting that the inductive signal might be incomplete, not strong enough or that only high-CO(2) conditions might be not sufficient for the cell stage to proceed to the formation of sexually active gametes. However, these gametogenesis-related proteins and/or hydroxyproline-rich glycoproteins may have novel roles outside the cell under high-CO(2) conditions.