Short-Flagella Mutants of Chlamydomonas reinhardtii

Six short-flagella mutants were isolated by screening clones of mutagenized Chlamydomonas for slow swimmers. The six mutants identify three unlinked Mendelian genes, with three mutations in gene shf-1, two in shf-2 and one in shf-3. shf-1 and shf-2 have been mapped to chromosomes VI and I, respectively. Two of the shf-1 mutations have temperature-sensitive flagellar-assembly phenotypes, and one shf-2 mutant has a cold-sensitive phenotype. shf shf double mutants were constructed; depending on the alleles present they showed either flagellaless or short-flagella phenotypes. Phenotypic revertants of shf-1 and shf-2 mutants were isolated, and certain of them were found to carry extragenic suppressors, some dominant and some recessive. We suspect that the shf mutations affect components of a specific flagellar size-control system, the existence of which has been suggested by a variety of physiological experiments.     

Properties of Phosphoglycolate Phosphatase from Chlamydomonas reinhardtii and Anacystis nidulans

The levels of activity of 2-phosphoglycolate phosphatase in the green algae, Chlamydomonas reinhardtii and Chlorella vulgaris, were in the range of 37 to 60 micromoles per milligram chlorophyll per hour and in the blue-green algae, Anacystis nidulans and Anabaena variabilis were 204 to 310 micromoles per milligram chlorophyll per hour. The activity in each species was similar regardless of whether the algae were grown with air or 5% CO₂ in air. The enzyme purified 530-fold from Chlamydomonas was stable, had a broad pH optimum between 6 and 8.5, and was specific for the hydrolysis of P-glycolate with a K_m of 23 micromolar. The enzyme purified 18-fold from Anacystis was labile, had a sharp pH optimum at 6.3, and was also specific for P-glycolate with a K_m of 94 micromolar. The molecular weight of the enzyme from Chlamydomonas was estimated to be 92,000 by gel filtration.

The phosphatase from both sources required a divalent cation for activity. The Chlamydomonas enzyme was most effectively activated by Co²⁺, but was also activated by Mg²⁺ (K_a = 30 micromolar), Mn²⁺, and Zn²⁺. The Anacystis enzyme was most effectively activated by Mg²⁺ (K_a = 140 micromolar), and was also activated by Co²⁺ and Mn²⁺, but not by Zn²⁺. Anions were also required for maximum activity of the enzyme from both sources. The Chlamydomonas enzyme was activated about 2- to 3-fold by chloride (K_a = 140 micromolar), bromide, nitrate, bicarbonate (K_a = 600 micromolar) and formate. The Anacystis enzyme was activated over 10-fold by chloride (K_a = 870 micromolar), bromide, iodide, and nitrate, but was not activated by bicarbonate or formate.

The properties of the algal enzymes were similar to those previously reported for higher plants. The levels and kinetic properties of the enzyme seemed sufficient to account for the flux through the glycolate pathway that occurs in these algae. The phosphatase was not associated with the ribulose 1,5-bisphosphate carboxylase/oxygenase responsible for P-glycolate formation in the carboxysomes of Anacystis.

     

Complement-Mediated Cytolysis of Cell-Wall Mutants of Chlamydomonas reinhardtii

Treatment of the cell wall-less mutant CW 15 of Chlamydomonasreinhardtii with human serum leads to a marked increase of thecell volume, followed by an irreversible cytolysis. Heat-inactivatedserum as a control reveals no cytotoxic effects on CW 15. Experimentswith C4-, properdin-, C3-, and factor H-depleted sera indicatethe alternative pathway of complement as being responsible forthe serum-mediated lysis. After immunofluorescence marking aswell as electromicroscopically after negative staining the membraneattacking complex of complement, C5b-9, could be demonstratedon the surface of CW 15. These results together with the observationthat cells of the wild-type strain 11-32c of C. reinhardtiiare not lysed by active serum suggest that only protoplastsof Chlamydomonas carry surface structures capable to activatethe alternative pathway of complement. In order to find out whether other cell wall mutants of C. reinhardtii,besides CW 15, can also activate the human complement system,we tested three strains each of the three known mutant categories.Strains CW 4, CW 9, and CW 19, representing category A, andstrains CW 3, CW 10, and CW 92, representing category C, andCW 8 and CW 18, accounting for category B, were cytolysed bynormal human serum. Only one type used in our experiments, CW20 of category B, resisted serum treatment, suggesting the needto redefine this category. ¹This paper is dedicated to Professor Dr. Andr? Pirson on theoccasion of his 80th birthday (Received December 1, 1989; Accepted April 5, 1990)     

Flagellar Morphology in Stumpy-Flagella Mutants of Chlamydomonas reinhardtii1

Sixteen new mutants of the biflagellate green alga Chlamydomonas reinhardtii with either stumpy-flagella or no flagella at all were examined by electron microscopy. Four of the mutants were found to carry short bulbous flagella containing amorphous electron-dense material which may represent unassembled flagellar protein. Basal bodies of normal ultrastructure were present in all mutants. Dikaryon dominance tests indicated that the stumpy mutations were recessive to wild-type in all cases tested. Stumpy mutations also conferred a measure of detergent resistance to Chlamydomonas, apparently by affecting the detergent-solubility of the flagellar membrane.     

Isolation and Characterization of Chemotaxis Mutants of Chlamydomonas reinhardtii

Zoospores of Chlamydomonas reinhardtii exhibit chemotaxis towards maltose, sucrose, xylose, mannitol, and ammonium. Ten independent mutants defective in chemotaxis towards sugars have been isolated. These mutants form five phenotypic classes. Genetic analysis of two mutant strains defective in chemotaxis to maltose (CHE1, CHE3) and two mutant strains defective in chemotaxis to sucrose (CHE2, CHE4) indicated that the defect in them depended on single nuclear recessive mutant alleles. Mutations mal1, mal2, suc1, and suc2 represent four chemotactic loci that are unlinked to the marker mt located on the linkage group VI. Four loci are unlinked to each other. These observations suggest that the mal and the suc loci do not constitute a spatially single functional group.     

Salt-Sensitive Mutants of Chlamydomonas reinhardtii Isolated after Insertional Tagging

We describe the isolation of salt-sensitive Chlamydomonas reinhardtii mutants by insertional mutagenesis using the nitrate reductase (Nit1) gene. The plasmid pMN24, containing Nit1, was used for transformation of 305CW15 (nit1 cw15 mt+), and transformants were selected for complementation of the nit- phenotype. From 6875 nit+ colonies, four transformants (S4, S18, S46, and S66) were isolated that exhibited both Na+ and Li+ sensitivity (sod-), and another transformant (S33) was selected that exhibited sensitivity to Li+ but not Na+ (lit-) based on relative growth comparisons with the wild-type strain. S33, S46, and S66 were no more growth inhibited by sorbitol than was 305CW15. In comparison, S4 and S18 exhibited substantial growth inhibition in medium supplemented with sorbitol. Genetic analyses indicated that the salt-sensitive mutants were each defective in a single recessive gene. The mutant genes in S4 (sod1), S33 (lit1), and S66 (sod3) are linked to a functional copy of Nit1 and are presumably tagged with a pMN24 insertion.     

野生型莱茵衣藻及其不同突变株的抗NaCl能力检测

检测莱茵衣藻的2种野生型CC-124、CC-125和15个不同突变株对NaCl抗性的结果表明,野生型品系CC-124和CC-125对NaCl的抗性达到260 mmo1·L-1,其中叶绿素b缺失的cbn1-48mt 和cbn1-48mt-基因突变株品系对NaCl最为敏感(即对100mmo1·L-1以上浓度的NaC1表现敏感).用紫外线照射诱变法,对野生型品系CC-124进行诱导,初步筛选出对150mmol·L-1NaCl敏感的突变株6个,对350mmol·L-1NaCl有抗性的突变株2个.     

Altered Flagellar Size-Control in shf-1 Short-Flagella Mutants of Chlamydomonas reinhardtii

Two allelic Mendelian mutations which confer a short flagella phenotype were used to explore flagellar size control in Chlamydomonas reinhardtii. When mutant/wild type quadriflagellate dikaryon cells were constructed, their two short flagella rapidly grew out to near wild type length. The kinetics of elongation suggest that the flagellar assembly process is not intrinsically self-limiting as a number of otherwise attractive models for size control require. Instead, we suggest that there exists a cellular machinery dedicated to flagellar size control and that the short-flagella mutations alter the machinery in some as yet unknown way. One of the mutants shows temperature-sensitive flagellar assembly, and both are flagellaless in acetate media. Genetic analysis indicates that the temperaturesensitive, acetate-sensitive, and short-flagella phenotypes have a common genetic basis. The responsible gene has been named shf-1, and it has been mapped to chromosome VI, approximately 5 map units from the centromere.     

Metabolite profiling of Chlamydomonas reinhardtii under nutrient deprivation

A metabolite profiling technique for Chlamydomonas reinhardtii cells for multiparallel analysis of low-molecular weight polar compounds was developed. The experimental protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity, and process large sample quantities. As a result of the rapid sampling, extraction, and analysis by gas chromatography coupled to time-of-flight mass spectrometry, more than 800 analytes from a single sample could be measured, of which more than 100 could be identified. Analyte responses could be determined mostly with ses less than 10%. Wild-type cells of C. reinhardtii strain CC-125 subjected to nitrogen-, phosphorus-, sulfur-, or iron-depleted growth conditions develop highly distinctive metabolite profiles. Individual metabolites undergo marked changes in their steady-state levels. Compared to control conditions, sulfur-depleted cells accumulated 4-hydroxyproline more than 50-fold, whereas the amount of 2-ketovaline was reduced to 2% of control levels. The contribution of each compound to the differences observed in the metabolic phenotypes is summarized in a quantitatively rigorous way by principal component analysis, which clearly discriminates the cells from different growth regimes and indicates that phosphorus-depleted conditions induce a deficiency syndrome quite different from the response to nitrogen, sulfur, or iron starvation.     

Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtii: Evidence for RNA Polymerase Mutants

The inhibitory activities of amatoxins on the growth of Chlamydomonas reinhardtii have been determined using a convenient assay based upon incubation in multiwell tissue culture plates followed by turbidimetric estimates of growth on a multiwell plate reader. Values for the inhibitory dosage at which growth is 50% of untreated culture (ID₅₀) of 5.4, 6.6, and 5.6 micromolar were obtained for α-amanitin, O-methyl-α-amanitin, and amaninamide, respectively. Treatment of liquid cultures with 1 microgram per milliliter N-methyl-N′ -nitro-N-nitrosoguanidine followed by growth in agar pour tubes containing 25 micromolar α-amanitin led to the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID₅₀ values from 36 micromolar to greater than 200 micromolar. Two lines completely resistant to inhibition by 200 micromolar α-amanitin provided partially purified RNA polymerase activities that were 160-fold and 5600-fold more resistant to inhibition than the analogous enzyme activity from the wild-type strain. These studies provide evidence that Chlamydomonas reinhardtii does not contain significant activity capable of inactivating α-amanitin and that this amatoxin may be used to select for RNA polymerase mutants.     

Mutants of Arabidopsis thaliana Capable of Germination under Saline Conditions

Three mutant strains of Arabidopsis thaliana var Columbia were selected for their ability to germinate in elevated concentrations of NaCl. They were not more tolerant than wild type at subsequent development stages. Wild-type strains could not germinate at concentrations > 125 mM NaCl. Two of mutant strains, RS17 and RS20, could withstand up to 225 mM, whereas RS19 was resistant to 175 mM. The RS mutants could also germinate under even lower osmotic potentials imposed by high concentrations of exogenous mannitol (550 mM), whereas the effects of elevated levels of KCl, K2SO4, and LiCl were similar among the mutants and wild type. Therefore, the mutants are primarily osmotolerant, but they also possess a degree of ionic tolerance for sodium. Sodium and potassium contents of seeds exposed to high salinities indicated that the NaCl-tolerant mutants absorbed more of these respective cations during imbibition. These higher internal concentrations of potassium and sodium could contribute to the osmotic adjustment of the germinating seeds to the low osmotic potential of the external medium. Genetic analysis of F1 and F2 progeny of outcrosses suggest that the salt-tolerant mutations are recessive and that they define three complementation groups.     

Cr6+胁迫对莱茵衣藻光合作用的影响

以莱茵衣藻(Chlamydomonas reinhardtii)为研究材料,采用氧电极和快速叶绿素a荧光诱导动力学方法研究了不同浓度和时间Cr6+处理对其光合作用的影响.结果表明:当Cr6+浓度大于40 μmol/L时,莱茵衣藻细胞数逐渐下降,而藻细胞变大;表观光合速率成为负值,呼吸作用随Cr6+处理浓度的增加先上升后下降至对照水平;莱茵衣藻有活性放氧复合体比例随Cr6+处理浓度的增加逐渐降低,80 μmol/L Cr6+处理3 d时已下降至13.72%;光合驱动力(DFABS)随Cr6+浓度增加逐步下降,并以DFφPo在DFABS的下降中的贡献最大.研究发现,重金属Cr6+胁迫显著影响莱茵衣藻的光合作用,而对呼吸作用则影响较小;Cr6+主要通过损伤供体侧的放氧复合体以及阻断QA至QB的电子传递而抑制光系统Ⅱ的功能;莱茵衣藻光系统Ⅱ对Cr6+处理比较敏感且存在着多个作用位点,并首先影响反应中心光能捕获效率,其次影响反应中心的活性,最后影响QA-之后的电子传递.     

Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation

The Chlamydomonas reinhardtii starch-less mutant, BAF-J5, was found to store lipids up to 65% of dry cell weight when grown photoheterotrophically and subjected to nitrogen starvation. Fourier transform infrared spectroscopy was used as a high-throughput method for semi-quantitative measurements of protein, carbohydrate and lipid content. The fatty acids of wild-type and starch mutants were identified and quantified by gas chromatography mass spectrometry. C. reinhardtii starch mutants, BAF-J5 and I7, produce significantly elevated levels of 16:0, 18:1(Δ9), 18:2(Δ9,12) and 18:3(Δ9,12,15) fatty acids. Long-chain saturated, mono- and polyunsaturated fatty acids were found under nitrogen starvation. Oleosin-like and caleosin-like genes were identified in the C. reinhardtii genome. However, proteomic analysis of isolated lipid bodies only identified a key lipid droplet associated protein. This study shows it is possible to manipulate algal biosynthetic pathways to produce high levels of lipid that may be suitable for conversion to liquid fuels.     

Chemoresponses of Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.     

Characterization of New Strains of Nonphotosynthetic Mutants of Chlamydomonas reinhardtii III. Photosystem II-Related Thylakoid Proteins in Five Mutants and Double Mutants

PS II-enriched particles of the wild type, of three mutantsand of two double mutants of Chlamydomonas reinhardtii wereanalyzed by lithium dodecylsulfate polyacrylamide gel electrophoresisat 4°C. The mutant Pg 27 was devoid of light-harvestingChl-protein complex (CP) CP II, but had normal cytochrome b-559and displayed all wild type photochemical activities. The mutantFl 50 lacked a pool of cytochrome b-559 photooxidizable at 77K but was able to photooxidize a second pool at 293 K in thepresence of FCCP; it showed some weak PS II activity. The mutantFl 39 lacked both these cytochrome b-559 pools and did not displayany PS II activity. The double mutants Fl 39 Pg 28 and Fl 50Pg 27 had defects similar to those of their respective parentsFl 39 or Fl 50 but, in addition, they were devoid of Chi b andof CP II. In these four mutants having impaired PS II function,five proteins of M_r=50,000, 47,000, 33,000, 27,000 and 19,000were totally (Fl 39, Fl 39 Pg 28) or partly (Fl 50, Fl 50 Pg27) missing. The first two of these proteins corresponded tothe apoproteins of CP III and IV. These results pointed out a strong correlation between thesefive proteins, cytochrome b-559 and PS II primary photochemistry.In mutation and cross experiments, these five PS II-associatedproteins and cytochrome b-559 appeared to be linked characterscontrolled by nuclear gene(s), but they behaved independentlyof CP II. (Received January 24, 1983; Accepted July 20, 1983)     

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.     

Cardiolipin of Chlamydomonas reinhardtii 137

The fatty acids of cardiolipin from the phototrophic green alga Chlamydomonas reinhardtii 137⁺ have been quantitatively analysed. Comparison is made at the molecular level between the cardiolipin of Chlamydomonas and that of higher plant tissue.     

Bioflocculant produced by Chlamydomonas reinhardtii

Bioflocculants of Chlamydomonas reinhardtii were investigated under axenic conditions. C. reinhardtii was found to produce significant amounts of bioflocculants. Flocculating activity by C. reinhardtii began in the linear phase of growth and continued until the end of the stationary phase. The highest flocculating efficiency of the culture broth was 97.06%. The purified C. reinhardtii bioflocculant was composed of 42.1% (w/w) proteins, 48.3% carbohydrates, 8.7% lipids, and 0.01% nucleic acid. The optimum condition for bioflocculant production of C. reinhardtii was as follows: under temperature of 15°C to 25°C, pH 6–10 and illumination of 40–60 μmol photons m^?2 s^?1. The bioflocculants produced by C. reinhardtii showed maximum activity in pH ranges from 2 to 10. The flocculating activity was significantly enhanced by the addition of CaCl₂ as a co-flocculant at an optimal concentration of 4.5 mM.     

Regulation of electron-transport pathways in cells of Chlamydomonas reinhardtii under stress conditions

The kinetics of interactions between electron-transport pathways in the thylakoid membrane was examined. A mathematical model was proposed to describe the kinetics of redox transitions in photosystem II, proton concentration changes in the chloroplast stroma, and the plastoquinone pool reduction due to photosynthetic and chlororespiratory pathways. A kinetic mechanism is considered that redirects electron flows between photosynthetic and chlororespiratory pathways in response to the increased NADPH content under mineral deficiency. According to the simulation model, the electron transport flows via different routes are switched over in a stepwise manner. The results of numerical simulations are qualitatively consistent with experimental data for Chlamydomonas reinhardtii cells subjected to mineral deprivation.