Induction and synthesis of tubulin during the cell cycle and life cycle of Chlamydomonas reinhardi

Two types of tubulin induction are observed in Chlamydomonas reinhardi. One is elicited by flagellar detachment and the other occurs as a normal event of the vegetative cell cycle. In the former case, a strong and extensive induction of tubulin synthesis occurs following deflagellation of cells in all phases of the life cycle [vegetative, gametic, and (early) zygotic]. Synthesis is initiated in all three cell types within 15 min after deflagellation. In gametic and zygotic cells, tubulin synthesis so induced accounts for 15 to 20% of the total protein synthesis during the 1-hr peak period of tubulin production. The ability to support both tubulin synthesis and flagellar regeneration is lost in zygotes at 1.5 hr after the initiation of zygotic development. This alteration represents one of several dramatic shifts in the programming of protein synthesis that occur during the first 4 hr of zygotic differentiation in C. reinhardi. The second (i.e., cell cycle-dependent) type of induction is observed in synchronously growing vegetative cells at ～1.5–2 hr prior to cytokinesis. Tubulin synthesis, in this case, persists at relatively high levels (～5% of the total protein synthesis) for the next 9 hr, i.e., through the entire period of cell division to a time just before the liberation of fully flagellated daughter cells at hr 20 of the cell cycle. Changes in the programming of protein synthesis, and of tubulin synthesis in particular, are discussed in relation to specific physiological and cytological transitions that occur during the growth and differentiation of C. reinhardi.     

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     

Gametic differentiation in Chlamydomonas reinhardtii. III. Cell wall lysis and microfilament-associated mating structure activation in wild- type and mutant strains

Cell fusion between mating type plus (mt+) and minus (mt-) gametes of Chlamydomonas reinhardtii is analyzed structurally and subjected to experimental manipulation. Cell wall lysis, a necessary prelude to fusion, is shown to require flagellar agglutination between competent gametes; glutaraldehyde-fixed gametes ("corpses") of one mating type will elicit both agglutination and cell wall lysis in the opposite mating type, whereas nonagglutinating impotent (imp) mutant strains are without effect. The fusion process is mediated by a narrow fertilization tubule which extends from the mt+ gamete and establishes contact with the mt- gamete. Formation of the tubule requires the "activation" of a specialized mating structure associated with the ml+ cell membrane; activation causes microfilaments to polymerize from the mating structure into the growing fertilization tubule. Mating structure activation is shown to depend on gametic flagellar agglutination; isoagglutination mediated by the lectin concanavalin A has no effect. Gametes carrying the imp-l mt+ mutation are able to agglutinate but not fuse with mt- cells; the imp-l gametes are shown to have structurally defective mating structures that do not generate microfilaments in response to gametic agglutination.     

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi.

Allophanate lyase can be induced by urea or acetamide 20-40-fold within 4 h in NH4 + -deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to the light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

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.     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were grown synchronouslyunder a 12 hr light-12 hr dark regime. Time courses of nucleardivision, chloroplast division, "apparent cytokinesis" and zoosporeliberation were followed during the vegetative cell cycle inthe synchronous culture. Liberation of zoospores occurred atabout 23–24 hr after the beginning of the light periodat 25°C. Four zoospores were produced per mother cell underthe conditions used. At lower temperatures, the process of zoosporeliberation as well as length of the cell cycle was markedlyprolonged, but the number of zoospores produced per mother cellwas approximately the same. At different light intensities,lengths of the cell cycle were virtually the same, while thenumber of zoospores liberated was larger at higher rather thanat lower light intensities. During the dark period, nuclear division, chloroplast divisionand apparent cytokinesis took place, in diis order, and proceededless synchronously than did the process of zoospore liberation.When the 12 hr dark period was replaced with a 12 hr light periodduring one cycle, the time of initiation as well as the durationof zoospore liberation was litde affected in most cases, whereasnuclear division, chloroplast division and apparent cytokinesiswere considerably accelerated by extended illumination. Whenalgal cells which had been exposed to light for 24 hr were furtherincubated in the light, zoospore liberation started much earlierand proceeded far less synchronously, compared with that under12 hr light-12 hr dark alternation. (Received October 12, 1970; )     

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.     

Nutritional control of sexuality in Chlamydomonas reinhardi

1. Cells of Chlamydomonas reinhardi grown in the light or dark on standard medium require an additional exposure to light in the absence of a nitrogen source, in order to become sexually active. As the culture ages, the light requirement decreases. 2. This light requirement is a function of nitrogen depletion, as shown by the observation that cells from cultures grown to maturity on a low nitrogen medium in the light or in the dark, have no additional light requirement for zygote formation. The withholding of no other component of the medium has this effect. 3. In cells requiring light for zygote formation, the light can be supplied before the mating types are mixed, indicating that light is required, not for mating per se, but for the conversion of vegetative cells to gametes. 4. Gametes can be dedifferentiated to the vegetative state by any nitrogen compound which the cells can use for growth; and by further exposure to light in the absence of a nitrogen source, these vegetative cells can again become gametic. 5. Cells grown at different nitrogen levels become gametic at widely different cell concentrations of nitrogen and carbon and C/N ratios. 6. It is postulated that the role of light in gametic differentiation is indirect, providing by photosynthesis, energy for the mating process and carbohydrates to tie up excess nitrogenous reserves; and that the concentration of some particular nitrogen fraction or compound determines whether or not gametic differentiation is initiated.     

Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi

Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique.     

Chlamydomonas reinhardtii: duration of its cell cycle and phases at growth rates affected by temperature

Synchronized cultures of the green alga Chlamydomonas reinhardtii were grown photoautotrophically under a wide range of environmental conditions including temperature (15–37°C), different mean light intensities (132, 150, 264 μmol m⁻² s⁻¹), different illumination regimes (continuous illumination or alternation of light/dark periods of different durations), and culture methods (batch or continuous culture regimes). These variable experimental approaches were chosen in order to assess the role of temperature in the timing of cell division, the length of the cell cycle and its pre- and post-commitment phases. Analysis of the effect of temperature, from 15 to 37°C, on synchronized cultures showed that the length of the cell cycle varied markedly from times as short as 14 h to as long as 36 h. We have shown that the length of the cell cycle was proportional to growth rate under any given combination of growth conditions. These findings were supported by the determination of the temperature coefficient (Q ₁₀), whose values were above the level expected for temperature-compensated processes. The data presented here show that cell cycle duration in C. reinhardtii is a function of growth rate and is not controlled by a temperature independent endogenous timer or oscillator, including a circadian one.     

Ethyl methanesulfonate: an effective mutagen in Chlamydomonas reinhardi

Summary The effectiveness of ethyl methanesulfonate (EMS) as a mutagen inChlamydomonas reinhardi has been demonstrated in two different systems: reversion to prototrophy of an argine-requiring strain and forward mutation to auxotrophy of the wild-type strain. In these experiments, the toxic effect of EMS was very low. With certain batches of EMS, an unusually high toxicity was observed. In this case, the lethal effects seem to be attributable to the presence of minor impurities in EMS.Chargé de Recherches au Fonds National de la Recherche Scientifique.     

Gametic Differentiation of Chlamydomonas reinhardtii: Control by Nitrogen and Light

Gametic differentiation of the unicellular green alga Chlamydomonas reinhardtii proceeds in two steps controlled by the extrinsic signals nitrogen deficiency and light. Nitrogen deprivation induces the differentiation of vegetative cells to sexually immature pregametes. A light signal is required to convert the pregametes to gametes. Both signals are also required for the maintenance of mating competence. Two converging signal transduction chains are proposed to control gamete formation. For the differentiation of pregametes to gametes, a fluence rate-dependent reaction, requiring continuous irradiation, is suggested by photobiological experiments.