Gametic differentiation in Chlamydomonas reinhardtii. I. Production of gametes and their fine structure

Gametogenesis in Chlamydomonas reinhardtii has been studied in mating-type plus cells utilizing several different culture conditions, all of which are shown to depend on the depletion of nitrogen from the medium, and the fine structure of gametes prepared under these conditions has been compared by using thin sections of fixed materials. We document alterations in ribosome levels, in chromatin morphology, in starch levels, in the organization of chloroplast membranes, and in the appearance of nuclear envelope and endoplasmic reticulum membranes during gametogenesis. We also noted the acquisition of two new organelles: a mating structure (Friedman, L., A. L. Colwin, and L. H. Colwin. 1968. j. cell Sci. 3:115-128; goodenough, U. W., and R. L. Weiss. 1975. J. Cell Biol. 67:623-637), and Golgi-derived vesicles containing a homogeneous material. We chart the time course of these morphological changes during synchronous gametogenesis. We note that many of these changes may represent adjustments to nitrogen starvation rather than direct features of gametic differentiation, and we also document that cells can differentiate so that they survive conditions of nitrogen starvation for many weeks after they become gametes. We conclude that metabolic alterations, the acquisition of mating ability, and the preparation for long-term survival are all elicited in this organism by nitrogen withdrawal, and we discuss how the various structural alterations observed in this study may relate to these three interrelated avenues of cellular differentiation.     

Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction

A structural and biochemical study is presented concerning the agglutination of gametic flagella, the initial step in the mating reaction of Chlamydomonas reinhardtii. An alteration in the distribution of the intramembranous particles revealed by freeze-fracturing of flagella membranes is shown to accompany gametic differentiation in both mating types. The isolation and electrophoretic analysis of flagellar membranes and mastigonemes are reported; no electrophoretic differences can be detected when the membrane or mastigoneme glycoproteins from vegative and gametic cells are compared, nor when glycoproteins from the two mating types are compared, and no novel polypeptides are present in gametic preparations. The membrane vesicles, after they are freed of mastigonemes by sedimentation through a discontinuous sucrose gradient, are extremely active as an isoagglutinin, indicating a direct involvement of the membrane in the mating reaction.     

Gametic differentiation in Chlamydomonas reinhardtii: light dependence and gene expression patterns

Gametes of Chlamydomonas reinhardtii synthesize numerous proteins not observed in vegetative cells and vice versa. Gametogenesis induced changes in gene expression were confirmed by SDS-PAGE of in vitro translation products using total RNA from gametes and vegetative cells. Vegetative cells and gametes thus represent two cell types with distinct patterns of gene expression. The generation of mature gametes from liquid cultures of asynchronously growing vegetative cells was dependent on light. This light requirement could not be substituted for by an organic source of energy and carbon, indicating that light serves as a signal in gametogenesis. The light signal was shown to become effective only after preincubation in nitrogen-free medium. This delayed competence for light indicates that the two external signals — nitrogen-starvation and light —may function in sequence. Execution of the light dependent step in gamete formation required cytoplasmic protein synthesis and RNA synthesis.Abbreviations CAM chloramphenicol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSII photosystem II - TAP Tris acetate phosphate - TMP Tris minimal phosphate This paper is dedicated by C. F. Beck to Professor John L. Ingraham, teacher and friend, on the occasion of his 65th birthday     

Comparative analysis of the biogenesis of photosystem II in the wild- type and Y-1 mutant of Chlamydomonas reinhardtii

Expression of the genes of the photosystem II (PSII) core polypeptides D1 and D2, of three proteins of the oxygen evolving complex of PSII and of the light harvesting chlorophyll a/b binding proteins (LHCP) has been compared in wild-type (wt) and in the y-1 mutant of Chlamydomonas reinhardtii. Since wt, but not y-1 cells produce a fully developed photosynthetic system in the dark, comparison of the two has allowed us to distinguish the direct effect of light from the influence of plastid development on gene expression. The PSII core polypeptides and LHCP are nearly undetectable in dark-grown y-1 cells but they accumulate progressively during light induced greening. The levels of these proteins in wt are the same in the light and the dark. The amounts of the proteins of the oxygen evolving complex do not change appreciably in the light or in the dark for both wt and y-1. Steady state levels of chloroplast mRNA encoding the core PSII polypeptides remain nearly constant in the light or the dark and are not affected by the developmental stage of the plastid. Levels of nuclear encoded mRNAs for the oxygen evolving proteins and of LHCP increase during light growth in wt and y-1. In contrast to wt, synthesis of LHCP proteins is not detectable in y-1 cells in the dark but starts immediately after transfer to light, indicating that LHCP synthesis is controlled by a light-induced factor or process. While the rates of synthesis of D1 and D2 are immediately enhanced by light in wt, this increase occurs only after a lag in y-1 and thus must be dependent on an early light-induced event in the plastid. These results show that the biosynthesis of PSII is affected by light directly, by the stage of plastid development, and by the interaction of light and events associated with plastid development.     

Ammonium Ions Control Gametic Differentiation and Dedifferentiation in Chlamydomonas reinhardtii

Effects of various nitrogen-containing compounds on gameticdifferentiation and dedifferentiation in Chlamydomonas reinhardtiiwere studied. Vegetative cells grown either non-synchronouslyor synchronously differentiated to gametes within 8 h in NH₄Cl-freemedium. Addition of arginine did not interfere with gameticdifferentiation although arginine was utilized by the gametesfor the progression of the cell cycle. Fully differentiatedgametes dedifferentiated rapidly to vegetative cells upon feedingwith     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

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)     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO₄) and oxidizing agents (NaClO₂) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)₂ inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.Abbreviations CSW chlorite-soluble cell-wall fraction - ICW insoluble cell-wall fraction - PSW salt-soluble fraction - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii is a metalloprotease and digests the sodium perchlorate-insoluble component of cell wall

Chlamydomonas lytic enzyme of the cell wall, which is released during agglutination of gametes of opposite mating types, has been characterized as a metalloprotease. The purified enzyme contains zinc. Removal of zinc with EDTA results in an inactive, metal-free apoenzyme, and Co2+ restores the activity most effectively. Among various protease inhibitors of microbial origin, pepstatin A, chymostatin, antipain, leupeptin, and E-64 do not inactivate the enzyme, whereas phosphoramidon causes a complete loss of lytic activity. Cysteine, histidine, aspartic acid, and glutamic acid also inhibit the activity. The lytic enzyme splits casein and RNase A into several polypeptides of lower molecular masses. To determine which polypeptides of the cell wall are sensitive to the lytic enzyme, we first separated the intact cell walls into sodium perchlorate-soluble and -insoluble components, treated them with enzyme, and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. We conclude that only 2 of 16 polypeptides are digested by exposure to the enzyme and that the sensitive polypeptides belong to the salt-insoluble component of the cell wall. The mechanism of cell wall digestion with the lytic enzyme is discussed.     

Increase in calcium triggers mating structure activation in Chlamydomonas eugametos

For mating Chlamydomonas eugametos gametes to fuse with their partners, they must first lyse part of the anterior cell wall and protrude their mating structures. These responses can be artificially induced by compounds that raise the Cai level, viz. InsP3, A23187, TFP and ethanol. We conclude that calcium should be considered with cAMP to be involved in signal transduction during C. eugametos mating.     

Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii

The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg²⁺ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface.     

Genetic analysis of mating type differentiation in Paramecium tetraurelia. III. A mutation restricted to mating type E and affecting the determination of mating type

In P. tetraurelia each cell is determined to express only one of the two complementary mating types, O and E. This determination is under cytoplasmic control and seems to be achieved only by the commitment or noncommitment to the expression of mating type E. All the previously known mutations affecting the differentiation of mating type prevent the expression of the E mating type (O-restricted mutations) without affecting the determination process. An E-restricted mutation was obtained: mtF^E. Its phenotypic properties indicate that the mutation affects the determination process itself. When an O cell becomes mtF^E/mtF^E it acquires the E mating type and an E-determining cytoplasm. We propose that this constitutive determination for the E mating type is due to the inefficiency of a factor which is normally active in an O cell. This factor would act like a repressor and stabilize the E functions under an inactive state.     

Gene expression in visceral endoderm: a comparison of mutant and wild- type F9 embryonal carcinoma cell differentiation

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.     

Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium.

In Chlamydomonas reinhardtii, cadmium induces reduction of growth, reduction of chlorophyll content, and lethality. The toxicity was higher in a cell wall-deficient strain than in the wild type. By growing the cells on agar medium containing cadmium at concentrations inducing high lethality, stable resistant clones were isolated. The resistance was due to a nuclear mutation (cadAR) which probably preexisted in the wild-type cell population, as suggested by the fluctuation test. A double mutant (cadAR cadBR) was selected on media containing higher concentrations of cadmium. The cadBR mutation, which is unlinked to cadAR, determines a resistance intermediate between the CadAR mutant and the wild-type strain. Both cadAR and cadBR mutations are partially dominant.     

Biohydrogen production using mutant strains of Chlamydomonas reinhardtii: The effects of light intensity and illumination patterns

Biohydrogen production from microalgae still remains to be discussed and examined more specifically, given that it is one of the most important energy carriers possessing environmental-friendly and sustainable characteristics. Although microalgae species capable of biohydrogen production do exist, Chlamydomonas reinhardtii is considered to be one of the most promising eukaryotic H₂ producers, and can serve as a model organism for such studies. Unfortunately, even if the metabolic basis and environmental conditions for this process are well defined, the sustainability of biohydrogen production is not straightforward. At this point, genetic engineering tools must be efficacious in order to enable mutant strains to reach desired amounts of biohydrogen. In this study, different light intensities, illumination patterns and Chlamydomonas strains such as CC124 and D1 protein mutant strains (D240, D239-40, D240-41) were investigated for the production of biohydrogen. The results showed that an increase in the light intensity shortened the lag phase of hydrogen production. With some minor differences, biohydrogen production was also found to be affected by the illumination pattern. On the other hand, maximum biohydrogen production was reached with a double-deletion mutant strain of D239-40, which attained a total production of 490 ± 10 mL L⁻¹ hydrogen and was followed by the other double-deletion mutant D240-41 that attained a total production of 388 ± 10 mL L⁻¹.     

Conservation in structure of TOC1 transposons from Chlamydomonas reinhardtii.

TOC1 transposons from Chlamydomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOC1 transposons were identified by restriction mapping on Southern blots. The variation in size of an internal MluI fragment defines two subclasses of TOC1 elements. Full-length cloned members of each subclass of TOC1 element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp are responsible for forming the two subclasses of TOC1 elements.     

The cell wall of Chlamydomonas reinhardtii gametes: Composition,structure and autolysin-mediated shedding and dissolution

The cell walls of Chlamydomonas gametes are multilayered structures supported on frameworks of polypeptides extending from the plasma membrane. The wall-polypeptide catalogue reported by Monk et al. (1983, Planta 158, 517–533) and extended by U.W. Goodenough et al. (1986, J. Cell Biol. 103, 405–417) was re-evaluated by comparative analysis of mechanically isolated cell walls purified from several strains. The extracellular locus of wall polypeptides was verified by in vivo iodogen-catalysed iodination and by autolysin-mediated elimination of the bulk of these polypeptides from the cell surface. Three (w15, w16, w17) and possibly four (w14) polypeptides were located to the most exterior aspect of the wall because of their susceptibility to Enzymobeadcatalysed iodination and their retention by a cell-wall-less mutant. The composition of shed walls stabilised with ethylenediaminetetraacetic acid during natural mating and kinetic analysis of the dissolution of walls purified from a bald-2 mutant demonstrated the rapid and specific destruction of polypeptide w3. Differential solubilisation of wall polypeptides occurred after loss of w3. Wall dissolution, characterised by the generation of fishbone structures from the W2 layer, gave as many as four additional polypeptides. Charged detergents and sodium perchlorate extracted a comparable range of polypeptides at room temperature from mechanically isolated walls, i.e. components of the W4–W6 layers, hot sodium dodecyl sulphate solubilised framework polypeptides, while reducing agent was required to solubilise the W2 layer. A model of wall structure is presented.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Mating type specific induction of cell wall lytic factor by agglutination of gametes in Chlamydomonas reinhardtii

When mt⁺ and mt^– gametes of Chlamydomonas reinhardtiiwere mixed, shedding of cell walls took place in both matingtypes during massive agglutination and/or pairing. This wascaused by a cell wall lytic factor that had been induced byflagellar agglutination and excreted into the medium by cellsconcurrently with their cell wall release. When glutaraldehyde-fixed gametes and isolated flagella of onemating type caused isoagglutination of live gametes of the othermating type, the live mt⁺ gametes induced the lytic factor andshed their walls, whereas none of the live mt^– did this.The cell walls of mt^– gametes were lost only when thelytic factor, which had been excreted by mt⁺ gametes into themedium, acted from the outside. These data imply that mt⁺ gametesare responsible for the induction of the lytic factor by agglutination,which acts on cell walls of both mating types either endogenouslyor exogenously. (Received February 28, 1978; )