Two iron-responsive promoter elements control expression of FOX1 in Chlamydomonas reinhardtii

FOX1 encodes an iron deficiency-induced ferroxidase involved in a high-affinity iron uptake system. Mutagenesis analysis of the FOX1 promoter identified two separate iron-responsive elements, FeRE1 (CACACG) and FeRE2 (CACGCG), between positions -87 and -82 and between positions -65 and -60, respectively, and both are needed for induced FOX1 expression under conditions of iron deficiency.     

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.     

Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach.

A real-time, sensitive, and simple assay for detection and quantification of adenosine triphosphate sulfurylase (ATP:sulfate adenylytransferase, EC 2.7.7.4) activity has been developed. The method is based on detection of ATP generated in the ATP sulfurylase reaction between APS and PPi by the firefly luciferase system. For the Saccharomyces cerevisiae ATP sulfurylase, the concentrations of APS and PPi at the half-maximal rate were found to be about 0.5 and 7 microM, respectively. The assay is sensitive and yields linear response between 0.1 microU and 50 mU. The method can be used for monitoring and quantification of recombinant ATP sulfurylase activity in Escherichia coli lysate, as well as for detection of the activity during different purification procedures.     

Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability.

A saturable and temperature-dependent copper uptake pathway has been identified in Chlamydomonas reinhardtii. The uptake system has a high affinity for copper ions (Km approximately 0.2 microM) and is more active in cells that are adapted to copper deficiency than to cells grown in a medium containing physiological (submicromolar to micromolar) copper ion concentrations. The maximum velocity of copper uptake by copper-deficient cells (169 pmol h-1 10(6) cells-1 or 62 ng min-1 mg-1 chlorophyll) is up to 20-fold greater than that of fully copper-supplemented cells, and the Km (approximately 2 x 10(2) nM) is unaffected. Thus, the same uptake system appears to operate in both copper-replete and copper-deficient cells, but its expression or activity must be induced under copper-deficient conditions. A cupric reductase activity is also increased in copper-deficient compared with copper-sufficient cells. The physiological characteristics of the regulation of this cupric reductase are compatible with its involvement in the uptake pathway. Despite the operation of the uptake pathway under both copper-replete and copper-deficient conditions, C. reinhardtii cells maintained in fully copper-supplemented cells do not accumulate copper in excess of their metabolic need. These results provide evidence for a homeostatic mechanism for copper metabolism in C. reinhardtii.     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.     

Gametic differentiation in Chlamydomonas reinhardtii: light dependence and gene expression patterns

Gametes of Chlamydomonas reinhardtii synthesize numerous proteins not observed in vegetative cells and vice versa. Gametogenesis induced changes in gene expression were confirmed by SDS-PAGE of in vitro translation products using total RNA from gametes and vegetative cells. Vegetative cells and gametes thus represent two cell types with distinct patterns of gene expression. The generation of mature gametes from liquid cultures of asynchronously growing vegetative cells was dependent on light. This light requirement could not be substituted for by an organic source of energy and carbon, indicating that light serves as a signal in gametogenesis. The light signal was shown to become effective only after preincubation in nitrogen-free medium. This delayed competence for light indicates that the two external signals — nitrogen-starvation and light —may function in sequence. Execution of the light dependent step in gamete formation required cytoplasmic protein synthesis and RNA synthesis.Abbreviations CAM chloramphenicol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSII photosystem II - TAP Tris acetate phosphate - TMP Tris minimal phosphate This paper is dedicated by C. F. Beck to Professor John L. Ingraham, teacher and friend, on the occasion of his 65th birthday     

Lipidomic Analysis of Chlamydomonas reinhardtii under Nitrogen and Sulfur Deprivation

Chlamydomonas reinhardtii accumulates lipids under complete nutrient starvation conditions while overall growth in biomass stops. In order to better understand biochemical changes under nutrient deprivation that maintain production of algal biomass, we used a lipidomic assay for analyzing the temporal regulation of the composition of complex lipids in C. reinhardtii in response to nitrogen and sulfur deprivation. Using a chip-based nanoelectrospray direct infusion into an ion trap mass spectrometer, we measured a diversity of lipid species reported for C. reinhardtii, including PG phosphatidylglycerols, PI Phosphatidylinositols, MGDG monogalactosyldiacylglycerols, DGDG digalactosyldiacylglycerols, SQDG sulfoquinovosyldiacylglycerols, DGTS homoserine ether lipids and TAG triacylglycerols. Individual lipid species were annotated by matching mass precursors and MS/MS fragmentations to the in-house LipidBlast mass spectral database and MS2Analyzer. Multivariate statistics showed a clear impact on overall lipidomic phenotypes on both the temporal and the nutrition stress level. Homoserine-lipids were found up-regulated at late growth time points and higher cell density, while triacyclglycerols showed opposite regulation of unsaturated and saturated fatty acyl chains under nutritional deprivation.     

Changes in gene expression patterns during the sexual life cycle of Chlamydomonas reinhardtii

Significant differences in membrane fluidities, expressed as fluorescence anisotropies, are demonstrated between embryogenic (E) and non-embryogenic (NE) cell lines when cells in suspension culture are removed from auxin. Cells of an E and NE cell line of Asclepias tuberosa were grown for 21 days either with or without 2, 4-dichlorophenoxy acetic acid (2,4-D), cultures were sampled at various intervals and protoplast membrane (hydrophobic interiors) was labeled with 1, 6 diphenyl-1, 3, 5-hexatriene (DPH). No differences between cultures with and without 2,4-D were detected in the NE line. In contrast the E line rapidly developed differences in membrane fluidity over time. Such clear differences in the responses of E and NE lines in membrane fluidity indicated that this parameter could be a good predictor and marker for embryogenesis. Eight suspension cell lines of Asclepias and 2 of Daucus carota were tested. After 2 days on medium without auxin, every E cell line exhibited a positive change in anisotropy and became embryogenic, whereas NE cell lines exhibited much lower positive changes or even negative changes in anisotropy and never underwent embryogenesis. Such changes have been consistent in all cell lines tested and represent a marker for embryogenicity in suspension cell lines before morphological change becomes apparent after removal from auxin. Basic molecular membrane changes in embryogenesis are likely to be common among different culture systems and understanding them could be a major step in removing barriers to regenerating plants from cultured material.     

Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii.

We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.     

Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron

Heterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5′ and 3′ sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation-independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.     

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

Summary A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2–3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.