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.     

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.     

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.     

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.     

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.     

Periplasmic Carbonic Anhydrase Structural Gene (Cah1) Mutant in Chlamydomonas reinhardtii

To survive in various conditions of CO₂ availability, Chlamydomonas reinhardtii shows adaptive changes, such as induction of a CO₂-concentrating mechanism, changes in cell organization, and induction of several genes, including a periplasmic carbonic anhydrase (pCA1) encoded by Cah1. Among a collection of insertionally generated mutants, a mutant has been isolated that showed no pCA1 protein and no Cah1 mRNA. This mutant strain, designated cah1-1, has been confirmed to have a disruption in the Cah1 gene caused by a single Arg7 insert. The most interesting feature of cah1-1 is its lack of any significant growth phenotype. There is no major difference in growth or photosynthesis between the wild type and cah1-1 over a pH range from 5.0 to 9.0 even though this mutant apparently lacks Cah1 expression in air. Although the presence of pCA1 apparently gives some minor benefit at very low CO₂ concentrations, the characteristics of this Cah1 null mutant demonstrate that pCA1 is not essential for function of the CO₂-concentrating mechanism or for growth of C. reinhardtii at limiting CO₂ concentrations.     

The Metabolic Status Drives Acclimation of Iron Deficiency Responses in Chlamydomonas reinhardtii as Revealed by Proteomics Based Hierarchical Clustering and Reverse Genetics

Iron is a crucial cofactor in numerous redox-active proteins operating in bioenergetic pathways including respiration and photosynthesis. Cellular iron management is essential to sustain sufficient energy production and minimize oxidative stress. To produce energy for cell growth, the green alga Chlamydomonas reinhardtii possesses the metabolic flexibility to use light and/or carbon sources such as acetate. To investigate the interplay between the iron-deficiency response and growth requirements under distinct trophic conditions, we took a quantitative proteomics approach coupled to innovative hierarchical clustering using different “distance-linkage combinations” and random noise injection. Protein co-expression analyses of the combined data sets revealed insights into cellular responses governing acclimation to iron deprivation and regulation associated with photosynthesis dependent growth. Photoautotrophic growth requirements as well as the iron deficiency induced specific metabolic enzymes and stress related proteins, and yet differences in the set of induced enzymes, proteases, and redox-related polypeptides were evident, implying the establishment of distinct response networks under the different conditions. Moreover, our data clearly support the notion that the iron deficiency response includes a hierarchy for iron allocation within organelles in C. reinhardtii. Importantly, deletion of a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), which is induced under low iron based on the proteomic data, attenuates the remodeling of the photosynthetic machinery in response to iron deficiency, and at the same time stimulates expression of stress-related proteins such as NDA2, LHCSR3, and PGRL1. This finding provides evidence that the coordinated regulation of bioenergetics pathways and iron deficiency response is sensitive to the cellular and chloroplast metabolic and/or redox status, consistent with systems approach data.The green alga Chlamydomonas reinhardtii has an enormous metabolic versatility (1) and possesses the flexibility to grow in the presence of different carbon sources. It may use carbon dioxide (CO₂) for photoautotrophic, acetate for heterotrophic, and both carbon sources for mixotrophic growth. In this alga CO₂ is fixed via the Calvin Benson Bassham cycle (2), while acetate can be taken up, converted to acetyl-CoA, and enter the glyoxylate cycle where it may be incorporated into C4 acids (3). In addition to the use of acetate as a source of energy and carbon backbone for biosynthetic processes, acetate can control respiration and photosynthesis in conjunction with the light intensity and CO₂ availability (4–6). Moreover, acclimation responses to iron- and copper-deficiencies significantly vary in photoautotrophic versus heterotrophic conditions (7–10), indicating that the metabolic status of the cells influence overall cellular acclimation responses.Transition metals like copper, manganese, and iron possess the ability to donate and accept electrons, making these metals suitable cofactors in enzymes that catalyze redox reactions. In particular, iron is used as a cofactor in numerous biochemical pathways and is therefore an essential nutrient. Cells require relatively high levels of iron because it is present in heme-, iron-sulfur and other proteins that function in respiratory and photosynthetic energy transducing. Correspondingly, in eukaryotic cells, the mitochondrion is a major iron-utilizing compartment. It is well established that iron is transported into mitochondria for heme synthesis and iron-sulfur cluster assembly. This is required for the formation of a functional respiratory electron transport machinery (11). Therefore, mitochondrial metabolism in mammals, fungi and plants is significantly affected under iron deficiency, as demonstrated by a number of studies (12–14). In plants, the chloroplasts are a primary target of iron deficiency. Changes in chloroplast structure, photosynthetic capacity and the composition of thylakoid membranes have been described for plants deprived of iron (15–21).Plants have devised various strategies for acquiring iron (22). Generally, iron deficiency leads to the activation of the iron uptake systems in photosynthetic organisms. For example, the accumulation of the ferroxidase, a component of the high affinity iron uptake system in C. reinhardtii, is very rapidly enhanced when iron becomes limiting (23). Inactivation of IRT1, the most prevalent Fe²⁺ transporter in Arabidopsis thaliana leads to a dramatic iron deficiency that is reflected by chlorosis (24–26). Despite the evolution of elaborate iron-uptake mechanisms in plants, iron deficiency-induced chlorosis remains a major agricultural problem (27, 28).The global impact of iron deficiency on photosynthetic productivity has been also shown in vast ocean regions, which are severely limited for iron (29, 30). Generally, one can conclude that photosynthesis in the oceans and on land can occur in environment where iron availability is restricted.Photosystem I (PSI) is a prime target of iron deficiency as it contains 12 atoms of iron per core complex. In algae, the degradation of PSI is also linked to remodeling of PSI-associated light-harvesting antenna (LHCI) (31–33). Cyanobacteria respond to iron deficiency by degradation of light harvesting phycobilisomes (34) and induction of the “iron-stress-induced” gene isiA. The ISIA protein, which has significant sequence similarity with CP43, a chlorophyll a-binding protein of photosystem II (PSII; (35, 36), forms a ring of 18 molecules around a PSI trimeric reaction center, as shown by electron microscopy (37, 38). The overall reorganization of the PSI complex from 900 kDa into 1.7 MDa complex highlights the large adaptive nature of the cellular response to iron deficiency, which helps to optimize the architecture of the photosynthetic apparatus to conditions in which iron is a limiting factor.The marine diatom Thalassiosira oceanica shows a remarkable retrenchment of cellular metabolism and remodeling of bioenergetic pathways in response to iron availability (39). Low iron triggers a reduction in the level of iron-rich photosynthetic proteins while iron-rich mitochondrial proteins are preserved. Furthermore, iron deprivation causes a remodeling of the photosynthetic machinery resulting in the adjustment of light energy use to an overall decline in the level of photosynthetic electron transport complexes (39). These responses, reported for green algae such as C. reinhardtii (31, 40, 41), are important for minimizing photo-oxidative stress and optimizing photosynthetic function. As observed for T. oceanica, under conditions of low iron availability (in the presence of organic carbon) a hierarchy of iron allocation responses in C. reinhardtii result in the down-regulation of iron-rich photosynthetic complexes while iron-rich mitochondrial complexes remain stable (41). Notably, under photoautotrophic and mixotrophic conditions C. reinhardtii displays distinct iron deprivation responses, suggesting that the cell''s response to iron deficiency is also dependent on trophic conditions (7–9). Thus bioenergetics pathways are remodeled in response to iron availability as well as to the type of carbon source available. Moreover, recent data has indicated that the regulation of iron-induced remodeling of the photosynthetic apparatus is linked to energy metabolism. Depletion of Proton Gradient Regulation Like1 protein (PGRL1) in C. reinhardtii has revealed a decreased efficiency of cyclic electron transfer under low iron conditions resulting in higher vulnerability toward iron deprivation (42).It was our aim to generate a more comprehensive picture of how the proteome of C. reinhardtii varies in response to low iron under distinct trophic conditions and how these changes compare with differences observed for cells grown under photoautotrophic and photoheterotrophic iron replete conditions. Quantitative proteomics in conjunction with a novel hierarchical clustering approach revealed information about the responses of C. reinhardtii to low iron conditions and the iron requirements of photoautotrophic growth. These analyses provide novel insights into the relationships between protein networks required for photosynthesis and iron deprivation-elicited stress responses; these studies are providing the knowledge required for modulating the level of available iron to improve the photosynthetic performance of plants (43, 44).     

SMART技术构建衣藻细胞的酵母双杂交文库

衣藻是用来研究植物光合作用和动物纤毛常用的模式生物.为了研究蛋白质之间的相互作用,利用SMART技术构建了衣藻的酵母双杂交文库.使用Trizol试剂提取鞭毛再生过程和光周期培养的细胞进入分裂前的衣藻细胞的总RNA,经过Oligotex纯化得到mRNA;应用SMART技术和LD-PCR合成双链cDNA,经过CHROMA SPIN TE-400柱子去除短片段的cDNA;cDNA和线性化载体pGADT7-Rec共转化酵母Y187构建酵母双杂交文库.库容达到3.0 × 106CFU,重组率为70％,插入片段平均长度为0.6 kb.以上结果说明该文库质量较好,能够通过筛选文库得到与目的蛋白互相作用的蛋白质,为寻找蛋白质的作用伴侣打下基础.     

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.     

Temperature-Sensitive, Photosynthesis-Deficient Mutants of Chlamydomonas reinhardtii

Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35°C. Whereas nonconditional mutants required acetate for growth at both 25 and 35°C, the conditional mutants have normal photoautotrophic growth at 25°C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35°C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35°C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebis-phosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis-deficient phenotypes was observed.     

Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. C hlamydomonas reinhardtii flagella use an asymmetric beat stroke, similar to a breast stroke, to propel cells forward. To generate the asymmetric beat stroke, dynein activity must be regulated both along the length and around the circumference of the flagella. If all dyneins were active at the same time, the flagella would exist in a state of rigor. The dyneins are located in two rows along the length of the doublet microtubules. The inner dynein arms are heterogeneous in composition with at least eight heavy chains and various intermediate and light chains arranged in an elaborate morphology that repeats every 96 nm (Kagami and Kamiya, 1992; Mastronarde et al., 1992). In contrast, the outer dynein arms are biochemically and morphologically homogeneous (Huang et al., 1979; Mitchell and Rosenbaum, 1985; Kamiya, 1988); each outer dynein arm contains three dynein heavy chains and 10 intermediate and light chains. The inner and outer arms appear to have different functions in the formation of the beat stroke; the inner arms generate the waveform of the beat stroke, whereas the outer arms provide additional force to the waveform (Brokaw and Kamiya, 1987).Previous workers had shown that dynein regulation is imposed, in part, by activities of the radial spokes and the central pair complex. Mutant strains that are missing or have altered radial spokes or central pair complexes are paralyzed even if they have a full complement of dyneins (Adams et al., 1981; Piperno et al., 1981). Many extragenic suppressors of this paralysis phenotype do not restore the missing structures, but rather suppress by altering either inner arm or outer arm region structures (Huang et al., 1982a ; Piperno et al., 1992; Porter et al., 1992, 1994). These data suggest that direct or indirect interactions exist between the dynein arms and the radial spokes or central pair complexes.Over 80 proteins in Chlamydomonas flagella are phosphorylated (Piperno et al., 1981), which makes dynein regulation by phosphorylation an attractive model. Hasegawa et al. (1987) showed that a higher percentage of demembranated axonemes reactivate with ATP after treatments that lower cAMP levels or inhibit cAMP-dependent protein kinase (cAPK)¹. In flagella from other organisms, cAMP has an opposite role (for reviews see Tash and Means, 1983; Tash, 1989). An increased frequency of reactivation also occurs after the NP-40–soluble components are extracted from the axonemes, which suggests that the cAPK, target phosphoproteins, and endogenous phosphatases are all integral axonemal components (Hasegawa et al., 1987). In quantitative sliding disintegration assays, the inner dynein arm activity of axonemes that are missing the radial spokes is increased in the presence of pharmacological or specific peptide inhibitors of cAPK (Smith and Sale, 1992; Howard et al., 1994). Reconstitution experiments with axonemes that are missing the radial spokes suggest that radial spokes normally function to activate the inner dynein arms by inhibiting a cAPK (Smith and Sale, 1992; Howard et al., 1994). It is not known if the cAPK directly phosphorylates inner dynein arm components or phosphorylates another axonemal component that then acts on the inner dynein arms (Howard et al., 1994).The f (originally called I1) inner arms are biochemically the best studied inner dynein arm complex. This complex is comprised of two dynein heavy chains and three intermediate chains of 140, 138, and 110 kD; it can be purified by sucrose density centrifugation (Piperno and Luck, 1981; Smith and Sale, 1991; Porter et al., 1992) or ion-exchange chromatography (Kagami and Kamiya, 1992). The purified complex has low ATPase activity and only rarely translocates microtubules in vitro (Smith and Sale, 1991; Kagami and Kamiya, 1992). Deep-etch EM of the purified f inner arm shows a two-headed complex that is connected to a common base by thin stalks (Smith and Sale, 1991). Longitudinal EM image analyses have shown that this complex is located just proximally of the first radial spoke in each 96-nm repeating unit (Piperno et al., 1990; Mastronarde et al., 1992). Mutations at three different loci (PF9/ IDA1, IDA2, and IDA3) result in the complete loss of the f complex (Kamiya et al., 1991; Kagami and Kamiya, 1992; Porter et al., 1992). The PF9/IDA1 locus encodes a dynein heavy chain that is believed to be one of the two heavy chains that are components of the f complex (Porter, 1996).We undertook a new approach to identify axonemal components involved in dynein regulation; we isolated and characterized mutant strains that were unable to perform phototaxis. In Chlamydomonas, phototaxis is a behavior by which cells orient to the direction of incident light. Light direction is detected by the eyespot, an asymmetrically located organelle, and a signal is transmitted to the flagella using voltage-gated ion channels (Harz and Hegemann, 1991). For cells to perform phototaxis, the waveforms of the two flagella are altered coordinately. The trans flagellum, which is located farther from the eyespot, beats with a larger front amplitude than the cis flagellum to turn the cell toward the light (Rüffer and Nultsch, 1991). It seemed likely that the alterations in the beat amplitudes needed for correct phototactic behavior could be caused by differential dynein regulation in the cis and trans flagella. Therefore, we hypothesized that there should be a class of phototactic mutant strains that is not able to perform phototaxis because of defects in the regulation of dyneins. Three of the eight phototactic mutant strains that we characterized had biochemical defects in the f class of inner dynein arms. One of these strains, pf9-4, was missing the entire f complex, and the other two strains, mia1-1 and mia2-1, exhibited a novel f class inner dynein arm biochemical phenotype. These observations suggest that the f inner dynein arm is a target for regulation during phototaxis.     

An anaplerotic role for mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii

Previous studies of the mitochondrial carbonic anhydrase (mtCA) of Chlamydomonas reinhardtii showed that expression of the two genes encoding this enzyme activity required photosynthetically active radiation and a low CO(2) concentration. These studies suggested that the mtCA was involved in the inorganic carbon-concentrating mechanism. We have now shown that the expression of the mtCA at low CO(2) concentrations decreases when the external NH(4)(+) concentration decreases, to the point of being undetectable when NH(4)(+) supply restricts the rate of photoautotrophic growth. The expression of mtCA can also be induced at supra-atmospheric partial pressure of CO(2) by increasing the NH(4)(+) concentration in the growth medium. Conditions that favor mtCA expression usually also stimulate anaplerosis. We therefore propose that the mtCA is involved in supplying HCO(3)(-) for anaplerotic assimilation catalyzed by phosphoenolpyruvate carboxylase, which provides C skeletons for N assimilation under some circumstances.     

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)     

An electro-optic monitor of the behavior of Chlamydomonas reinhardtii cilia

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.     

Visualization of microbodies in Chlamydomonas reinhardtii

In Chlorophycean algal cells, these organelles are generally called microbodies because they lack the enzymes found in the peroxisomes of higher plants. Microbodies in some algae contain fewer enzymes than the peroxisomes of higher plants, and some unicellular green algae in Chlorophyceae such as Chlamydomonas reinhardtii do not possess catalase, an enzyme commonly found in peroxisomes. Thus, whether microbodies in Chlorophycean algae are similar to the peroxisomes of higher plants, and whether they use a similar transport mechanism for the peroxisomal targeting signal (PTS), remain unclear. To determine whether the PTS is present in the microbodies of Chlorophycean algae, and to visualize the microbodies in Chlamydomonas cells, we examined the sub-cellular localization of green fluorescent proteins (GFP) fused to several PTS-like sequences. We detected GFP compartments that were spherical with a diameter of 0.3-1.0?μm in transgenic Chlamydomonas. Comparative analysis of the character of GFP-compartments observed by fluorescence microscopy and that of microbodies by electron microscopy indicated that the compartments were one and the same. The result also showed that the microbodies in Chlorophycean cells have a similar transport mechanism to that of peroxisomes of higher plants.     

Chloroplast Development in the Chloroplast Ribosome Deficient Mutant (y-1 ac-20) of Chlamydomonas reinhardtii

To study the participation of chloroplast protein synthesisduring the three phases [Matsuda (1974) Biochim. Biophys. Acta366:45] of the greening process in Chlamydomonas reinhardtiiy-1, the greening characteristics in the low-chloroplast ribosomemutant y-1 ac-20 were compared with those in the y-1. In thedouble mutant cells Chl synthesis proceeded with an extendedlag and without a second transition point. The development ofpotential for rapid Chl synthesis (P-factor formation) was alsodelayed. Furthermore, PS I activity increased significantly,whereas PS II activity developed very little during greeningof the double mutant cells. The results indicate that greeningin double mutant cells occurs with no apparent late phase. (Received November 26, 1984; Accepted February 25, 1985)     

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。     

衣藻生物制氢的研究进展

综述了利用衣藻生产氢气作为再生能源的研究进展。分别介绍了衣藻产氢的代谢机理、培养条件、衣藻氢化酶的特性以及利用分子生物学手段、生物信息学手段和生物工程技术提高衣藻生物制氢效率的方法,包括氢化酶的氧耐受性的改造、外源氢化酶基因的表达、影响衣藻产氢的关键基因的筛选、利用缺硫培养基和固定化培养方法提高氢气产量等。最后,还对利用衣藻生物制氢的可行性和经济性进行了分析,对其发展方向提出自己的看法。     

Photosynthetic efficiency of Chlamydomonas reinhardtii in attenuated, flashing light

As a result of mixing and light attenuation, algae in a photobioreactor (PBR) alternate between light and dark zones and, therefore, experience variations in photon flux density (PFD). These variations in PFD are called light/dark (L/D) cycles. The objective of this study was to determine how these L/D cycles affect biomass yield on light energy in microalgae cultivation. For our work, we used controlled, short light path, laboratory, turbidostat‐operated PBRs equipped with a LED light source for square‐wave L/D cycles with frequencies from 1 to 100 Hz. Biomass density was adjusted that the PFD leaving the PBR was equal to the compensation point of photosynthesis. Algae were acclimated to a sub‐saturating incident PFD of 220 µmol m^?2 s^?1 for continuous light. Using a duty cycle of 0.5, we observed that L/D cycles of 1 and 10 Hz resulted on average in a 10% lower biomass yield, but L/D cycles of 100 Hz resulted on average in a 35% higher biomass yield than the yield obtained in continuous light. Our results show that interaction of L/D cycle frequency, culture density and incident PFD play a role in overall PBR productivity. Hence, appropriate L/D cycle setting by mixing strategy appears as a possible way to reduce the effect that dark zone exposure impinges on biomass yield in microalgae cultivation. The results may find application in optimization of outdoor PBR design to maximize biomass yields. Biotechnol. Bioeng. 2012; 109: 2567–2574. © 2012 Wiley Periodicals, Inc.