A Mutant of Chlamydomonas reinhardtii with Reduced Rate of Photorespiration

Photorespiration rates under air-equilibrated conditions (0.04%CO₂ and 21% O₂) were measured in Chlamydomonas reinhardtii wild-type2137, a phosphoglycolate-phosphatase-deficient (pgp1) mutantand a suppressor double mutant (7FR2N) derived from the pgp1mutant. In both cells grown under 5% CO₂ and adapted air for24 h in the suppressor double mutant, the maximal rate of photorespiration(phosphoglycolate synthesis) was only about half of that ineither the wild type or the pgp1 mutant (18-7F) cells. In theprogeny, the reduced rate of photorespiration was accompaniedby increased photosynthetic affinity for inorganic carbon andthe capacity for growth under air whether accompanied by thepgp1 background or not. Tetrad analyses suggested that thesethree characteristics all resulted from a nuclear single-genemutation at a site unlinked to the pgp1 mutation. The decreasein photorespiration was, however, not due to an increase inthe CO₂/O₂ relative specificity of ribulose-1,5-bisphosphatecarboxylase/oxygenase of 7FR2N or of any other suppressor doublemutants tested. The relationship between the decrease in therate of photorespiration and the CO₂-concentrating mechanismis discussed. ³ Current address: Institute of Botany, Academy of Sciences,Patamdar Shosse, 40, Baku, 370073, Azerbaijan. ⁴ Current address: Department of Management and InformationScience, Jobu University, 270-1, Shinmachi, Tano, Gunma, 370-1393Japan.     

A Photorespiratory Mutant of Chlamydomonas reinhardtii

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO₂ concentration for photoautrophic growth in spite of the apparent induction of a functional CO₂ concentrating mechanism in air-adapted cells. In 2% O₂ the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O₂, photosynthetic O₂ evolution is severely inhibited in the mutant by preillumination in limiting CO₂, although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O₂ evolution measurements. Net CO₂ uptake is also inhibited when the cells are exposed to air (21% O₂, 0.035% CO₂, balance N₂) for longer than a few minutes. [¹⁴C]Phosphoglycolate accumulates within 5 minutes of photosynthetic ¹⁴CO₂ fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO₂ requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.     

Effect of Dim Light on the y-1 Mutant of Chlamydomonas reinhardtii

The y-1 mutant of Chlamydomonas reinhardtii tends to die or revert to wild type when grown in the dark for a long period of time. A small amount of white light (0.5 lux) enables the y-1 mutant to grow indefinitely in a “near dark” condition. Under this condition, the y-1 mutant is physiologically and ultrastructurally similar to the dark-grown y-1 yet remains genetically stable.     

Blue Light Induction of Carbonic Anhydrase Activity in Chlamydomonas reinhardtii

Blue light was specifically required for the induction of carbonicanhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancingeffect of blue light (460 nm) was saturated at energy fluencerate as low as 0.6-0.8 W/m². The wavelength dependency curvehad a peak at 460 nm with no effect at wavelengths above 510nm, thus showing the strong similarities to other blue lightresponses in microalgae. CA induction was strongly inhibitedby UV irradiation at 280 nm. Experiments with the flavin quencher,potassium iodide, suggested that flavin is somehow involvedin CA induction. ¹On leave from the Institute of Biological Sciences, Collegeof Arts and Sciences, University of the Philippines at Los Banos,4031 College, Laguna, Philippines. (Received August 29, 1988; Accepted November 26, 1988)     

Control of Gametic Differentiation and Activity by Light in Chlamydomonas reinhardtii

Laboratory strains of Chlamydomonas reinhardtii, which are descendantsof a 1945 isolate by G.M. Smith (Harris 1989), were dividedinto two groups depending upon whether the vegetative cellsrequire light to differentiate into gametes under ammonium ion-starvedconditions. Light-dependent (LD) strains were unable to becomegametes in the dark, while light-independent (LI) strains coulddo so. All the wild-type strains isolated recently from thefield showed light-dependency, suggesting that the LD-phenotypeis the wild-type. The LD-cells failed to acquire flagellar agglutinability,to accumulate cell body agglutinins, or to form mating structuresin the dark, but did so rapidly after transfer to light. Moreover,the light-induced LD-gametes, but not the Li-gametes, lost theirmating ability, cell body agglutinins, and mating structuresafter transfer to darkness, indicating that the LD-cells requirelight not only for gametic differentiation but also for maintenanceof gametic activity. (Received July 4, 1997; Accepted October 17, 1997)     

Light Inhibits Flagellation in a Chlamydomonas Mutant

We have isolated a new flagellar mutant in Chlamydomonas reinhardtii.When the mutant was cultured under the white fluorescent lamp({small tilde}4,800 lux), most cells had no flagella. However,when the cultures were put in the dark, flagellation occurred.Greater than 70% of the cells had flagella within 12–16h after the transfer. The flagellar morphology varied from "rod-shape"(same as the wild-type flagella) to "disk-shape". The disk-shapedflagella had the axonemes which were curved into a loop withinthe swollen membrane. Hence, this mutant is called loop-1. Light-inhibitionof flagellation was restored in the presence of 10^–5 MDCMU. The spectral dependency of the photo-inhibition of flagellation,determined using the Okazaki Large Spectrograph, showed maximaleffectiveness at 400–420 nm and 600–680 nm. Theseresults suggest that photosynthesis inhibits flagellation ofloop-1 cells. (Received July 27, 1989; Accepted January 29, 1990)     

Characterization of a Mutant of Chlamydomonas reinhardtii That Uses L-Methionine-S-Sulfoximine and Phosphinothricin as Nitrogen Sources for Growth

A strain of Chlamydomonas reinhardtii, named ARF-1, which grows with the glutamine synthetase (GS) inhibitor L-methionine-S-sulfoximine (MSX), has been isolated and characterized. Mutant ARF-1 is affected at a single and dominant gene, tentatively assigned to the allele msr-1-2. Neither the uptake of ammonia nor the two GS isoenzyme activities of the mutant were affected by MSX in vivo. GS activities, however, were fully abolished in vitro, thus suggesting that neither GS isoform was an altered enzyme resistant to the inhibitor. Resistance to MSX does not seem to be due to either a defect in a permease responsible for the transport of MSX or over-expression of GS activity, nor did we find an alternative enzymatic pathway for the assimilation of ammonium. Resistance was independent of the nitrogen source used and was strongly enhanced by the addition of acetate. Unlike the parental strain, mutant ARF-1 can degrade and utilize MSX as the sole nitrogen source for growth, which could account for the observed resistance. Thus, this mutant can be classified as a novel type of MSX-resistant mutant. This mutant can also use phosphinothricin, methionine sulfone, or methionine sulfoxide as the sole sources of nitrogen. This capability cosegregated in the genetic crosses and was also observed in all the diploids isolated. An MSX/[alpha]-ketoglutarate aminotransferase activity, not present in the parental strain 305, was detected in mutant ARF-1 cells. Therefore, we propose that the locus msr-1-2 either codes for this transaminase activity or its product gene is necessary to express this transaminase activity.     

Mutant strains of Chlamydomonas reinhardtii that move backwards only

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak- like projections found within the B-tubules of outer doublets 5 and 6.     

Dual Role of Methionine in the Biosynthesis of Diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii

Biosynthesis of the polar group of diacylglyceryl-O-4′-(N,N,N-trimethyl)homoserine (DGTS) was studied in intact cells of Chlamydomonas reinhardtii Dangeard. Among the three C₄ amino acids tested, only l-methionine could specifically inhibit the photosynthetic incorporation of [¹⁴C]NaHCO₃ into the polar group of DGTS. The radioactivity in l-[¹⁴C]methionine, which was labeled at either the C3 + C4, the C1, or the methyl carbon, was efficiently incorporated into the polar group of DGTS. These results suggest that the C₄ backbone and the S-methyl group of l-methionine are precursors to the C₄ backbone and the N-methyl groups of DGTS, respectively.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.     

Blue Light Regulation of Cell Division in Chlamydomonas reinhardtii

A delay in cell division was observed when synchronized cultures of the unicellular green alga Chlamydomonas reinhardtii growing under heterotrophic conditions were exposed to white light during the second half of the growth period. This effect was also observed when photosynthesis was blocked by addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Light pulses of 10 minutes were sufficient to induce a delay in cell division in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A delay in cell division was induced by blue light but not by illumination with red or far-red light. The equal intensity action spectrum revealed two peaks at 400 and 500 nm.     

Photoconversion of Photochlorophyllide in the y-1 Mutant of Chlamydomonas reinhardtii

Dark-grown y-1 mutant cells of Chlamydomonas reinhardtii accumulate protochlorophyllide (Pchlide) in both 635 nanometers (P635) and 650 nanometers (P650) forms. Plastids in these cells lack the normal thylakoid membrane structure except some remnants of membrane vesicles. Using difference spectrophotometry, P635 is shown to be photoconverted to chlorophyllide at 672 nanometers (C672) and P650 is photoconverted to C688 followed by a rapid shift to C672 (Shibata shift) and regeneration of P650. Some of the Pchlide is not photoconverted despite repeated illumination. Although P650 is destroyed by freezing and thawing, it is not transformed into P635. Freezing and thawing treatment also made Pchlide no longer photoactive.     

Effect of Chlorophyll-Protein Complex I Deficiency on the Physiological Character of a Chlamydomonas reinhardtii Mutant

In the mutant CC-1047 of Chlamydomonas reinhardtii, LDS-PAGE showed that the chlorophyll-protein complex I (CPI) is almost absent. The mutant could not grow in a culture medium without organic carbon source while the wild type (WT) C. reinhardtii grew quickly. When an organic carbon source was added into the culture medium, the mutant grew almost as well as WT. The rate of photosystem 1 (PS1) electron transport (DCPIPMV) and the rate of whole chain electron transport (H₂OMV) of chloroplasts of the CC-1047 mutant were both lower than those of WT. The photophosphorylation activity, photosynthetic O₂ evolution rate, and rate of NADP⁺ photoreduction of CC-1047 were also much lower than the activities of WT. There were some differences in ATPase activity between the mutant and WT. Two different activation ways were used to activate the latent ATPase using methanol and dithiothreitol (DTT) as activation substrate. More methanol and DTT were required for the mutant than WT to obtain the maximum activity. Thus the photosynthetic apparatus could not operate normally when CPI was absent because of the abnormal PS1 electron transport. Meanwhile, the other adjacent complexes of the thylakoid membrane, for example, ATP synthase complex, were slightly affected.     

Blue Light-Induced Lethality of a Cell Wall-Deficient Mutant of the Unicellular Green Alga Chlamydomonas reinhardtii

When synchronized cultures of a cell wall-deficient Chlamydomonasreinhardtii mutant strain were grown under heterotrophic conditionsand subsequently transferred to the light, a considerable decreaseof the cell number was observed during transition to the celldivision phase. Lethality of the wall-deficient cells was inducedby blue light, but not by red or far-red light, and could notbe prevented by addition of the photosystem II inhibitor DCMU.The light-induced lethality was found to be restricted to wall-deficientcells which were agitated by bubbling with filtered air or nitrogenor vigorously shaken during the transition to the cell divisionphase. Therefore, a (blue) light-induced sensitivity to anymechanical stress seems to be the cause for cell death. In heterotrophicallygrowing cultures of the Chlamydomonas wild-type, illuminationwith blue or white light did not cause a decrease of the cellnumber but only a delay of cell divisions. The latter effectwas also observed in case of the wall-deficient mutant. Bothblue light effects are observed during the transition to thecell division phase and can be induced during the same periodof the cell cycle. Furthermore, the (blue) light-induced lethalityof wall-deficient cells was found to be prevented when the transitionto the cell division phase was inhibited by addition of antibiotics.Therefore, we assume that there is a connection between theblue light-induced sensitivity to mechanical stress and theblue light-induced delay of cell divisions. (Received September 3, 1993; Accepted November 12, 1993)     

Chlamydomonas reinhardtii as a model system for pro-active herbicide resistance evolution research

Modern herbicides greatly contribute to world agricultural production but their sustainability is threatened by the widespread evolution of herbicide resistant weedy plant populations. Despite the commercial and scientific importance of resistance, there has not been an experimental model system for pro-actively evaluating the potential for herbicide resistance evolution. Here, utilizing the rapidly growing, unicellular photosynthetic microalgae Chlamydomona s reinhardtii (Dangeard), a ratchet protocol has been developed that solves the problem of maintaining both large populations and strong herbicide selection. The ratchet protocol is a progressive set of cycles, each cycle commencing with a population of approximately one million individuals apportioned amongst three herbicide doses for 14 days. Whenever the evolving population demonstrates growth across the three herbicide selection intensities, then the population ratchets to the next cycle of higher herbicide dose. Therefore, by always maintaining large populations under selection pressure, this system offers the opportunity for beneficial mutations to arise and be enriched. Using the well-characterized atrazine herbicide, the ratchet protocol resulted in rapid evolution of populations with different levels of resistance. This robust laboratory based Chlamydomonas system is proposed for application in establishing the respective propensity for resistance evolution to herbicides or other selecting agents.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 257–266.     

Acid Sensitivity of a Mutant of Lactococcus lactis subsp. lactis C2 with Reduced Membrane-bound ATPase Activity

Mutants with reduced membrane-bound ATPase activities were isolated from Lactococcus lactis subsp. lactis C2 as spontaneous neomycin-resistant mutants. Characteristics of the representative mutant, No. 1016–51, were compared with the parental strain in cultures using a jar fermentor with the pH controlled at various values. At pH 6.5, the fermentation patterns, i.e., glucose consumption, growth, and lactic acid production, of both strains appeared identical. At pH 4.5, however, the levels of growth, lactic acid production, and the amounts of lactic acid produced per cell after the culture for 24 h decreased to 60, 36, and 60% of the parental strain, respectively. During the cultures at pH 6.5, no differences were found in viabilities between both strains even after 80 h. On the other hand, at pH 4.0, the viable count of the strain No. 1016–51 in a 72-h culture decreased to less than 1% of that of the zero time, while the parental strain maintained its original viability. Therefore, it was concluded that the membrane-bound ATPase is essential for this organism to survive at low pH, probably through its function of proton pumping for maintaining cytoplasmic pH levels.