The lost neuromotor apparatus of Chlamydomonas: rediscovered

Early light microscopic studies of the biflagellate green alga Chlamydomonas revealed a fibrous system, the neuromotor apparatus, which appeared to link physically the flagellar apparatus to the cell nucleus. Following the development of the electron microscope, the existence of a neuromotor apparatus in Chlamydomonas was cast into doubt since it was not observed in studies carried out at ultrastructural resolution. Here we show, by indirect immunofluorescence, using monoclonal antibodies and electron microscopy employing refined specimen preparation and staining techniques, that the neuromotor apparatus of Chlamydomonas does indeed exist. The functional significance of this system is discussed in light of both historic proposals and recent experimental findings.     

Characterization of a Chlamydomonas reinhardtii gene encoding a protein of the DNA photolyase/blue light photoreceptor family

The organization and nucleotide sequence of a gene from Chlamydomonas reinhardtii encoding a member of the DNA photolyase/blue light photoreceptor protein family is reported. A region of over 7 kb encompassing the gene was sequenced. Northern analysis detected a single 4.2 kb mRNA. The gene consists of eight exons and seven introns, and encodes a predicted protein of 867 amino acids. The first 500 amino acids exhibit significant homology with previously sequenced DNA photolyases, showing the closest relationship to mustard (Sinapis alba) photolyase (43% identity). An even higher identity, 49%, is obtained when the Chlamydomonas gene product is compared to the putative blue-light photoreceptor (HY4) from Arabidopsis thaliana. Both the Chlamydomonas and the Arabidopsis proteins differ from the well characterized DNA photolyases in that they contain a carboxyl terminal extension of 367 and 181 amino acids, respectively. However, there is very little homology between the carboxyl terminal domains of the two proteins. A previously isolated Chlamydomonas mutant, phrl, which is deficient in DNA photolyase activity, especially in the nucleus, was shown by RFLP analysis not to be linked to the gene we have isolated. We propose this gene encodes a candidate Chlamydomonas blue light photoreceptor.     

PHYLOGENY OF CHLAMYDOMONAS (CHLOROPHYTA) BASED ON CLADISTIC ANALYSIS OF NUCLEAR 18S rRNA SEQUENCE DATA1

The genus Chlamydomonas Ehrenberg may contain as many as 450 described species. Morphological, physiological and molecular data show that variation among some Chlamydomonas species can he great, leading to speculation that multiple, generic-level lineages exist within this genus. The most recent systematic studies of Chlamydomonas have led to proposals of nine distinct morphological and 15 distinct sporangial autolysin groups. Partial sequences from the nuclear small subunit rRNAs from 14 Chlamydomonas species representing 12 autolysin and four morphological groups, and from three flagellates thought to he related to Chlamydomonas were determined in a phylogenetic study of relationships among these algae. Sequence comparisons among some Chlamydomonas species revealed differences comparable to the sequence divergence between soybeans and cycads. Cladistic analysis of the sequence data suggests that multiple lineages exist among species of Chlamydomonas. Some of these lineages represent alliances of both Chlamydomonas and non-Chlamydomonas taxa; thus, the current taxonomy does not reflect natural, or monophyletic, groups. Collectively, these lineages may represent distinct families or even orders.     

Cross-reconstitution of the extrinsic proteins and Photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea

Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

Cellular aggregation in Chlamydomonas (Chlorophyceae) is chimaeric and depends on traits like cell size and motility

How the variation in phenotypic traits like cell size and motility impacts predator-induced cellular aggregation is not known. Furthermore the genetic composition of cell groups in mixed populations of Chlamydomonas has not been investigated. An examination of these two questions will not only enhance our understanding of Chlamydomonas ecology, but also shed light on the primordial steps before integrated multicellular groups were established. Group living comes with viability and reproductive costs and it is not known how these are shared if groups are genetically heterogeneous. We observed that the natural predator Peranema trichophorum (Euglenoidea) induced clumping in Chlamydomonas. When co-cultured with P. trichophorum cells protected themselves by forming facultative groups (reverting back to a unicellular lifestyle once predators were removed). The dynamics of group formation in different Chlamydomonas species and strains correlated with cell size and swimming speed. Small or less motile strains aggregated more readily than large, fast-swimming ones. Interestingly, Chlamydomonas groups were both intra-species and inter-species chimaeric. This suggests that the predator-induced group formation in Chlamydomonas involved cells coming together rather than staying together and during aggregation cells showed little or no discrimination between self and non-self. These data demonstrate that the dynamics of cell aggregation, in unicellular volvocines at least, depends on phenotypic traits like cell size and motility and high genetic relatedness is not mandatory at this initial stage. These findings further our understanding of aggregation in mixed Chlamydomonas populations and have implications for understanding the very first steps on the road to simple multicellularity.     

Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales,Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov.

Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400–600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy—in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria—and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup “Pleiochloris,” was included in the genus Ixipapillifera as I. deasonii comb. nov.     

A fully automated and computerized system for simultaneous measurements of motility and phototaxis in Chlamydomonas

A fully automated and computerized method for simultaneous measurements of motility and phototaxis of unicellular flagellates is described. Both systems are directly coupled with a homocontinuous culture. The motility measuring apparatus is equipped with a video camera and recorder for simultaneous single cell behaviour studies. First results of studies on the effects of the phototaxis inhibitor sodium azide and the Ca²⁺ conducting ionophore A23 187 on motility and phototaxis of Chlamydomonas are reported and correlated with video observations. These results demonstrate that the described systems give informations of whether phototaxis or motility or both are inhibited by chemicals.     

The CO2-concentrating mechanism is absent in the green alga Coccomyxa: a comparative study of photosynthetic CO2 and light responses of Coccomyxa, Chlamydomonas reinhardtii and barley protoplasts

Photosynthesis was characterized for the unicellular green alga Coccomyxa sp., grown at low inorganic carbon (C_i) concentrations, and compared with Chlamydomonas reinhardtii, which had been grown so that the CO₂ concentrating mechanism (CCM) was expressed, and with protoplasts isolated from the C₃ plant barley (Hordeum vulgare). Chlamydomonas had a significantly higher C_i-use efficiency of photosynthesis, with an initial slope of the C_i-response curve of 0.7 mol(gChl)⁻¹ h⁻¹ mmol C_im⁻³)⁻¹, as compared to 0.3 and 0.23 mol(gChl)⁻¹ h⁻¹ (mmol C_im⁻³)⁻¹ for Coccomyxa and barley, respectively. The affinity for C_i was also higher in Chlamydomonas, as the half maximum rate of photosynthesis [K_0.5 (C_i)] was reached at 0.18 mol m⁻³, as compared to 0.30 and 0.45 mol m⁻³ for Coccomyxa and barley, respectively. Ethoxyzolamide (EZ), an inhibitor of the enzyme carbonic anhydrase (CA) and the CCM, caused a 17-fold decrease in the initial slope of the photosynthetic Cj-response curve in Chlamydomonas, but only a 1.5- to two-fold decrease in Coccomyxa and barley. The photosynthetic light-response curve showed further similarities between barley and Coccomyxa. The rate of bending of the curve, described by the convexity parameter, was 0.99 (sharp bending) and 0.81–0.83 (gradual bending) for cells grown under low and high light, respectively. In contrast, the maximum convexity of Chlamydomonas was 0.85. The intrinsically lower convexity of Chlamydomonas is suggested to result from the diversion of electron transport from carbon fixation to the CCM. Taken together, these results suggest that Coccomyxa does not possess a CCM and due to this apparent lack of a CCM, we propose that Coccomyxa is a better cell model system for studying C₃ plant photosynthesis than many algae currently used.     

THE FINE STRUCTURE OF THE FLAGELLAR APPARATUS OF CARTERIA1,2

The fine structure of the flagellar apparatus of 5 species of the green quadriflagellate alga Carteria is described. The 5 species can be morphologically separated into 2 groups on the bases of cell shape and ultrastructure of the pyrenoid and flagellar apparatus. Group I cells are spherical, possess many pyrenoid thylakoids, and retain a flagellar apparatus similar to that of Chlamydomonas reinhardi. The flagellar bases are oriented at approximately 90° to one another, have distal and proximal fibers, and are associated with 4 cruciately arranged microtubule bands. Cells of group II are ellipsoid, possess few pyrenoid thylakoids, and show a complex system of microtubule bands and sigmoid-shaped, electron dense rods which extend between opposite pairs of basal bodies. The basal bodies of group II cells are directed inward in a circular pattern rather than outward as in group I cells. Unlike Chlamydomonas, the distal fiber of the Carteria species is nonstriated. The proximal fiber is striated, and both distal and proximal fibers are composed of 60–80 Å diameter microfibrils.     

Localization of Pyrophosphatase and V-ATPase in Chlamydomonas reinhardtii

Microsomal membranes of Chlamydomonas reinhardtii possess PPase and V-ATPase activities. By immunogold labelling we have shown that H⁺-pyrophosphatase (PPase) is localized to membranes of lytic and contractile vacuoles of Chlamydomonas, in which the density of antigen in the latter is much higher. In addition, PPase is conspicuously present in trans cisternae and transpole elements of the Colgi apparatus. Such a distribution for PPase has hitherto not been reported. A positive in situ identification for PPase at the plasma membrane, including the flagellar membrane, was also made, and has also been confirmed by Western blotting and activity measurements on isolated plasma membranes. V-ATPase antisera which cross react with polypeptides of this transport complex from maize roots failed to recognize anything in Western blots of Chlamydomonas microsomal membranes. Thus immunogold labelling for V-ATPase was not possible with Chlamydomonas. On the other hand, surfaces of contractile vacuole membranes as revealed by deepetching were covered by conspicuous 9 ? 11.5 nm diameter smooth particles which had a central hole. These were very similar to those previously identified by Heuser et al., (1993) as the V,-head of V-ATPase in Dictyostelium contractile vacuoles. Another type of membrane image, designated “intermediate-sized vesicle”, was found associated with the contractile vacuole. It was characterized by densely-packed 6 ? 7.5nm diameter polygonal particles, which upon rotation analysis showed both 5- and 6-fold symmetries, also with a central hole. These particles are interpreted as representing either PPase complexes or the V₀ body of the V-ATPase in etched fractured membrane surfaces. We have incorporated these findings into a model of contractile vacuole function.     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase     

The flagellar root system of zoospores of the green algaChlorosarcinopsis (Chlorosarcinales) as compared withChlamydomonas (Volvocales)

The flagellar root system of zoospores in two species ofChlorosarcinopsis (C. minuta andC. spec.) has been studied in detail. The biflagellate zoospores show a cruciate root system, two of the four microtubular roots containing two microtubules, the other two four microtubules. The flagellar apparatus is otherwise identical with that ofChlamydomonas reinhardi as described byRingo (1967). Evidence is presented that the genusChlamydomonas is characterized by a bilateral symmetric root system (4-2-4-2) rather than a system with four equally numbered roots (i.e. 4-4-4-4). It is suggested that a root system with four identical cruciate roots is not present in any biflagellate algal cell. The taxonomic significance of cruciate root systems in green algae is discussed refering to the identical root systems ofChlorosarcinopsis andChlamydomonas.     

All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.

The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 × longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a β-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

     

Spring development of a Chlamydomonas population in Lake Nimetön,a small humic forest lake in southern Finland

Biomass development and vertical distribution of a Chlamydomonas population in a small humic forest lake was followed by daily sampling in May-June, 1984. Chlamydomonas dominated the phytoplankton spring bloom, forming 71% of the maximum phytoplankton biomass on 18 May. In early May the outflow rate was high and during the 24 hour period when the maximum rate of surface runoff was recorded (8–9 May), 43% of the Chlamydomonas biomass was flushed out of the lake, which delayed the onset of biomass increase. When surface runoff had slowed down Chlamydomonas biomass started increasing and during wax of the population most cells were < 10 µm in diameter. Population maximum lasted for one day (18 May) and there-after Chlamydomonas biomass decreased towards the end of the study. During wane of the population most cells were > 10 µm in diameter.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

The Antarctic psychrophiles Chlamydomonas spp. UWO241 and ICE-MDV exhibit differential restructuring of photosystem I in response to iron

Chlamydomonas sp. UWO241 is a psychrophilic alga isolated from the deep photic zone of a perennially ice-covered Antarctic lake (east lobe Lake Bonney, ELB). Past studies have shown that C. sp. UWO241 exhibits constitutive downregulation of photosystem I (PSI) and high rates of PSI-associated cyclic electron flow (CEF). Iron levels in ELB are in the nanomolar range leading us to hypothesize that the unusual PSI phenotype of C. sp. UWO241 could be a response to chronic Fe-deficiency. We studied the impact of Fe availability in C. sp. UWO241, a mesophile, C. reinhardtii SAG11-32c, as well as a psychrophile isolated from the shallow photic zone of ELB, Chlamydomonas sp. ICE-MDV. Under Fe-deficiency, PsaA abundance and levels of photooxidizable P700 (ΔA₈₂₀/A₈₂₀) were reduced in both psychrophiles relative to the mesophile. Upon increasing Fe, C. sp. ICE-MDV and C. reinhardtii exhibited restoration of PSI function, while C. sp. UWO241 exhibited only moderate changes in PSI activity and lacked almost all LHCI proteins. Relative to Fe-excess conditions (200 µM Fe²⁺), C. sp. UWO241 grown in 18 µM Fe²⁺ exhibited downregulation of light harvesting and photosystem core proteins, as well as upregulation of a bestrophin-like anion channel protein and two CEF-associated proteins (NdsS, PGL1). Key enzymes of starch synthesis and shikimate biosynthesis were also upregulated. We conclude that in response to variable Fe availability, the psychrophile C. sp. UWO241 exhibits physiological plasticity which includes restructuring of the photochemical apparatus, increased PSI-associated CEF, and shifts in downstream carbon metabolism toward storage carbon and secondary stress metabolites.