Induction of deflagellation by various local anesthetics in Chlamydomonas reinhardtii Dangeard (Chlamydomonadales,Chlorophyceae)

Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod‐like shape to a disk‐like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell‐wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1‐h EC₅₀) for flagellar excision are as follows: dibucaine (1.37 × 10^?5 M) < tetracaine (3.16 × 10^?5 M) < bupivacaine (4.25 × 10^?4 M) < procaine (2.02 × 10^?3 M) < lidocaine (3.61 × 10^?3 M). In all cases, Ca²⁺ depletion from the solution inhibited flagellar excision. However, Ca²⁺‐channel blockers, IP3 receptor antagonists, and inhibitors of phospholipase C did not prevent excision. We suggest that the local anesthetics induce flagellar excision by increasing the fluidity of the flagellar/cell membrane, thereby allowing extracellular Ca²⁺ to flow into the cell and cause flagellar excision.     

Characterization of the Eyespot Regions of "Blind"Chlamydomonas Mutants after Restoration of Photophobic Responses

Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC-2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash-induced photophobic responses to visible light stimuli to which wild-type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all-trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild-type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild-type C. reinhardtii to those of both mutants. As previously described, wild-type cells contain an eyespot consisting of 2–4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange-red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC-2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68-like mutants studied earlier by Morel-Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN-68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance. Consistent with these findings, several laboratories have shown that the full range of phototactic responses can be reconstituted in FN68 and CC-2359, but that negative phototaxis requires a significantly stronger light stimulus in the latter strain. We confirm the suggestion that the eyespot is not necessary for the photophobic response, and is not critical for the appropriate assembly and function of the photophobic response receptor in the membrane. Furthermore, the locus of reconstitution of the functional receptor is not the eyespot. Because of the definitive demonstration of the absence of the eyespot in CC-2359, however, the eyespot may play a role in negative phototaxis.     

A new 'paralyzed' flagella mutant, OC-10, in Chlamydomonas reinhardtii (Volvocales, Chlorophyceae) that can be reactivated with adenosine triphosphate

A new ‘paralyzed’ mutant. OC–10, was isolated in Chlamydomonas reinhardtii Dangeard. OC-10 cannot swim and generally shows very little flagellar movement. However, when OC-10 was demembranated, axonemal motility was reactivated in the presence of adenosine triphosphate (ATP) or adenosine diphosphate (ADP). The beating form of the reactivated axonemes was almost the same as that of the wild-type axonemes. Flagellar regeneration of OC-10 was slower than that of the wild-type. Electron microscopic examination showed no abnormality in OC-10 flagella, but SDS/PAGE revealed that mobility of a flagellar membrane protein was changed and a few bands disappeared in OC-10 flagella, When the mutant was crossed to wild-type to form temporary dikaryon cells with 4 flagella, OC-10 flagella did not regain motility. Tetrad analysis of crosses between OC–10 and wild-type demonstrated a 1:1 segregation on the basis of flagellar motility. From these results, we suppose that OC-10 may be limited in ATP availability inside the flagella, or altered in flagellar membrane proteins important for motility.     

Hybridization between Japanese and North American Chlamydomonas reinhardtii (Volvocales,Chlorophyceae)

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild‐type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type‐specific and nitrate reductase‐related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii.     

Genetic analysis of mutant clones of Chlamydomonas reinhardtii defective in potassium transport

Mutant clones of Chlamydomonas reinhardtii defective for potassium transport were isolated and characterized. Of the four genes identified, three –TRK1, TRK2 and TRK3– encode high-affinity transport functions, and one gene, HKR1, encodes a low-affinity transport function. Characterization of the potassium dependence of recombinants possessing two mutant trk alleles suggests that the protein products of TRK2 and TRK3 interact functionally, and that TRK1 may serve a regulatory function. The mutant clone defective for a low-affinity potassium transporter was isolated by mutagenizing trk2-1 cells, which lack a functional high-affinity transporter, and screening surviving cells for dependence on very high potassium concentrations. The hkr1 phenotype is expressed only in the presence of trk2-1. Received: 24 August 1998 / Accepted: 16 November 1998     

Chronological transition of mitochondrial morphology in Chlamydomonas reinhardtii (Chlorophyceae) poststationary phase growth1

In Chlamydomonas reinhardtii P. A. Dangeard, mitochondrial morphology has been observed during asexual cell division cycle, gamete and zygote formation, zygote maturation, and meiotic stages. However, the chronological transition of mitochondrial morphology after the stationary phase of vegetative growth, defined as the poststationary phase, remains unknown. Here, we examined the mitochondrial morphology in cells cultured for 4 months on agar plates to study mitochondrial dynamics in the poststationary phase. Fluorescence microscopy showed that the intricate thread‐like structure of mitochondria gradually changed into a granular structure via fragmentation after the stationary phase in cultures of about 1 week of age. The number of mitochondrial nucleoids decreased from about 30 per cell at 1 week to about five per cell after 4 months of culture. The mitochondrial oxygen consumption decreased exponentially, but the mitochondria retained their membrane potential. The total quantity of mitochondrial DNA (mtDNA) of cells at 4 months decreased to 20% of that at 1 week. However, the mitochondrial genomic DNA length was unchanged, as intermediate lengths were not detected. In cells in which the total mtDNA amount was reduced artificially to 16% after treatment with 5‐fluoro‐2‐deoxyuridine (FdUrd) for 1 week, the mitochondria remained as thread‐like structures. The oxygen consumption rate of these cells corresponded to that of untreated cells at 1 week of culture. This suggests that a decrease in mtDNA does not directly induce the fragmentation of mitochondria. The results suggest that during the late poststationary phase, mitochondria converge to a minimum unit of a granular structure with a mitochondrial nucleoid.     

Effects of Calcium Ions and of Calcium Channel Blockers on Galvanotaxis of Chlamydomonas reinhardtii

The effects of calcium ions and of the calcium channel blockers verapamil, diltiazem and nifedipine on galvanotaxis in Chlamydomonas have been investigated using a fully automated and computerized population system. Galvanotaxis is a function of the voltage applied to the cell population. However, the galvanotactic orientation also depends on the external calcium concentration. In a calcium-deprived nutrient medium which still contains 6 × 10^?7M calcium, galvanotactic orientation is about 20% of orientation at optimal calcium concentration of 10^?4 M at 9 V. The higher the external calcium concentration is, the lower is the voltage necessary for optimal galvanotactic orientation. The calcium channel blockers diltiazem and nifedipine likewise inhibit galvanotaxis of Chlamydomonas very specifically without impairing motility. Verapamil is effective, but also inhibits motility by causing detachment or shortening of the flagella. Nevertheless, inhibition of galvanotaxis by verapamil is not the only result of decreased motility, because the galvanotactic orientation is impaired to a greater extent than motility. The effectiveness of the three blockers tested in inhibiting galvanotaxis depends on the concentration and on the voltage applied. At 10^?5 M, verapamil causes maximal inhibition of galvanotaxis at 9 V. At increasing concentrations up to 10^?4 M, diltiazem inhibits galvanotaxis more strongly than the other blockers. If the voltage is varied at a constant blocker concentration of 2 × 10^?5 M, nifedipine causes maximal inhibition at 3 V–6 V, diltiazem at 9 V and verapamil above 12 V.     

Effects of doubled atmospheric CO2 concentration on the growth and photosynthesis of Chlamydomonas reinhardtii (Volvocales, Chlorophyceae)

The freshwater microalga, Chlamydomonas reinhardtii Dangeard, was cultured under 350 and 700 ppmv CO₂ to determine the impact of doubled atmospheric CO₂ concentration on its growth and photosynthesis. No significant difference was observed in the specific growth rate, photosynthetic efficiency, maximal net photo‐synthetic rate and light‐saturating point between the low and high CO₂ cultures. Both the low‐ and high‐CO₂‐grown cells showed reduced light‐dependent O₂ evolution rate and photochemical efficiency (F_v/F_m) owing to photoinhibition when exposed to high photon flux density. However, high‐CO₂‐grown cells were less photoinhibited, and showed better recovery in dim light or darkness during the initial period of the recovery process.     

Selective Inhibition of Flagellar Activity in Chlamydomonas by Nickel

Flagellar activity in the biflagellate chlorophyte Chlamydomonas reinhardtii is selectively inhibited by Ni²⁺ or by treatment with Ca²⁺-chelating agents. Inhibitions of swimming speed, geotaxis, phototaxis, and pattern swimming result from qualitative and quantitative losses in the activity of individual flagella and in the coordination of activity beween the 2 flagella of each cell. Addition of Ca²⁺ (a) prevents inhibition and (b) restores normal flagellar activity in inhibited cells. Mg²⁺ is partially effective in reversal of inhibition. Other ions do not cause similar inhibition or reversal of nickel inhibition. The characteristics of inhibition and reversal suggest that the prmary target for nickel is a component of the flagellar apparatus, and that this component uses Ca²⁺ to perform its normal function in the regulation of flagellar activity. A 2nd target for nickel is a Carequiring process specific to phototaxis (and not involved in the photophobic response).     

Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene

Efficient chloroplast transformation systems now available allow the manipulation of the evolutionarily highly conserved psbA gene in the eucaryotic organism Chlamydomonas reinhardtii. Two copies of this gene in the inverted repeat region of the chloroplast genome contain four large group I introns. To analyse possible functions of these introns and to generate a mutant for simplified psbA gene manipulations, a psbA cDNA fragment was introduced into a psbA deletion mutant using the biolistic transformation method. A transformant with no introns in the psbA gene has been obtained and represents the first example of the removal of a complete set of introns from a chloroplast gene. The newly generated strain is photosynthetically competent and contains no detectable recipient genome copies. The loss of all four introns appears to be phenotypically silent.     

Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae)

A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.     

莱茵衣藻Nfr-4突变株中类胡萝卜素含量的变化及其对藻生长的影响

文章对相同条件下培养的莱茵衣藻野生型CC-137和八氢番茄红素脱氢酶(phytoene desaturase,PDS)基因突变株Nfr-4的生长进行分析;并用反相高效液相色谱分析总有色类胡萝卜素以及叶绿素含量变化,结果表明两者生长的差异明显;Nfr-4突变株的单细胞叶绿素和总有色类胡萝卜素含量高于野生型CC-137的。     

Photosynthetic apparatus organization and function in the wild type and a chlorophyll b-less mutant of Chlamydomonas reinhardtii. Dependence on carbon source

 The assembly, organization and function of the photosynthetic apparatus was investigated in the wild type and a chlorophyll (Chl) b-less mutant of the unicellular green alga Chlamydomonas reinhardtii, generated via DNA insertional mutagenesis. Comparative analyses were undertaken with cells grown photoheterotrophically (acetate), photomixotrophically (acetate and HCO⁻ ₃) or photoautotrophically (HCO⁻ ₃). It is shown that lack of Chl b diminished the photosystem-II (PSII) functional Chl antenna size from 320 Chl (a and b) to about 95 Chl a molecules. However, the functional Chl antenna size of PSI remained fairly constant at about 290 Chl molecules, independent of the presence of Chl b. Western blot and kinetic analyses suggested the presence of inner subunits of the Chl a-b light-harvesting complex of PSII (LHCII) and the entire complement of the Chl a-b light-harvesting complex of PSI (LHCI) in the mutant. It is concluded that Chl a can replace Chl b in the inner subunits of the LHCII and in the entire complement of the LHCI. Growth of cells on acetate as the sole carbon source imposes limitations in the photon-use efficiency and capacity of photosynthesis. These are manifested as a lower quantum yield and lower light-saturated rate of photosynthesis, and as lower variable to maximal (F_v/F_max) chlorophyll fluorescence yield ratios. This adverse effect probably originates because acetate shifts the oxidation-reduction state of the plastoquinone pool, and also because it causes a decrease in the amount and/or activity of Rubisco in the chloroplast. Such limitations are fully alleviated upon inclusion of an inorganic carbon source (e.g. bicarbonate) in the cell growth medium. Further, the work provides evidence to show that transformation of green algae can be used as a tool by which to generate mutants exhibiting a permanently truncated Chl antenna size and a higher (per Chl) photosynthetic productivity of the cells. Received: 10 November 1999 / Accepted: 22 December 1999     

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae,Chlamydomonadales)

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.     

Pleiotropic mutants hypersensitive to heavy metals and to oxidative stress in Chlamydomonas reinhardtii

Insertional mutagenesis was used in Chlamydomonas reinhardtii to isolate original mutants hypersensitive to multiple drugs and physical agents. Out of 5200 transformants analyzed, 13 mutants belonging to seven phenotypic classes were isolated. Five were exclusively sensitive to cadmium and represented two loci. The other mutants were pleiotropic and presented a cross sensitivity to several (2--6) of the following agents: cadmium, copper, lead, paraquat, hydrogen peroxide, UVC and light. In all mutants analyzed, the hypersensitive phenotype was most probably due to a single mutational event. The sensitivity of several pleiotropic mutants to a broad range of physical and chemical agents suggests that the disrupted genes are involved in multiple stress responses.     

Transformation of CW-15 Mutant Cells of Chlamydomonas reinhardtii Dang. with pCTVHyg Plasmid

A pCTVHyg plasmid was constructed in a unicellular green alga Chlamydomonas reinhardtii Dang. by using the hygromycin phosphotransferase gene (hpt) as a selectable marker and the Escherichia coli transposon Tn5 under the early SV40 viral gene promoter. CW-15 mutant cells devoid of cell walls were transformed by electroporation in an electric field of 1 kV/cm and a pulse duration of 2 ms. A suspension density of 10⁶ cell/ml and the mid-logarithmic growth phase were the optimum conditions for transformation, producing up to 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ Hyg^R recipient cells. Exogenous DNA integrated in the nuclear genome of C. reinhardtii was steadily inherited in subsequent generations within at least a 8-month period; however, the Hyg^R trait manifestation was not stable. The comparative analysis of frequencies in codon usage in hpt and in the nuclear genes of C. reinhardtii significantly excluded the possibility that the bias in codon usage was the primary factor affecting foreign gene expression. The advantages of using theCW-15 mutant and the described selection system are discussed in the context of heterologous transformation of C. reinhardtii.     

Acutely induced cell mortality in the unicellular green alga Chlamydomonas reinhardtii (Chlorophyceae) following exposure to acrylic resin nanoparticles

Nanoparticles have unique properties that make them attractive for use in industrial and medical technology industries but can also be harmful to living organisms, making an understanding of their molecular mechanisms of action essential. We examined the effect of three different sized poly(isobutyl‐cyanoacrylate) nanoparticles (iBCA‐NPs) on the unicellular green alga Chlamydomonas reinhardtii. We found that exposure to iBCA‐NPs immediately caused C. reinhardtii to display abnormal swimming behaviors. Furthermore, after one hour, most of the cells had stopped swimming and 10%–30% of cells were stained with trypan blue, suggesting that these cells had severely impaired plasma membranes. Observation of the cyto‐ultrastructure showed that the cell walls had been severely damaged and that many iBCA‐NPs were located in the space between the cell wall and plasma membrane, as well as inside the cytosol in some cases. A comparison of three strains of C. reinhardtii with different cell wall conditions further showed that the cell mortality ratio increased more rapidly in the absence of a cell wall. Interestingly, cell mortality over time was essentially identical regardless of iBCA‐NP size if the total surface area was the same. Furthermore, direct observation of the trails of iBCA‐NPs indicated that the first trigger was their contact with the cell wall, which is most likely accompanied by the inactivation or removal of adsorbed proteins from the cell wall surface. Cell mortality was accompanied by the overproduction of reactive oxygen species, which was detected more readily in cells grown under constant light rather than in the dark.     

淀粉合成受限的莱茵衣藻的甘油酯酰基的变化

【目的】基于突变藻株本身属性和意义出发,考察在两种常用培养方式下莱茵衣藻淀粉突变株(CC-4326)与野生型藻株(CC-137)在甘油酯中酰基随生长的变化差异,为进一步认识莱茵衣藻突变株提供参考信息。【方法】分别在柱状鼓泡式反应器和摇瓶中培养CC-4326和CC-137,比较两株藻在正常培养和氮胁迫培养状态下甘油酯中酰基相对含量和其在甘油三酯(TAG)含量的差异。【结果】正常培养状态下,CC-4326和CC-137中多不饱和酰基C16:4和C18:3相对含量占总酰基45%左右,CC-4326在两种培养方式下这两个酰基含量及变化无差别,而CC-137在摇瓶中培养二者相对含量增加幅度和含量均高于反应器。缺氮条件下两种藻株积累TAG,但程度不同,CC-4326在反应器中培养TAG含量达到CC-137的1.5倍,在摇瓶中培养含量与CC-137无显著差异,两株藻的甘油酯和TAG中C18:1含量显著增加,CC-4326在反应器中培养C18:1增加幅度大于摇瓶,比摇瓶培养更快速积累TAG。而CC-137在摇瓶中培养TAG含量与反应器接近,单不饱和酰基增加幅度却高于反应器,表明CC-137在摇瓶中培养比反应器更利于积累TAG。最终,CC-4326在光生物反应器中缺氮培养实现了TAG 12倍的增加。【结论】通过对淀粉合成抑制,与CC-137相比,缺氮光生物反应器培养条件下,CC-4326能够实现TAG的高效积累。