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.     

Dissection of the Blue-Light-Dependent Signal-Transduction Pathway Involved in Gametic Differentiation of Chlamydomonas reinhardtii

Gametogenesis of the green alga Chlamydomonas reinhardtii may be viewed as a two-step process that is controlled by the environmental cues of nitrogen deprivation and blue light. Initiation of gametogenesis is induced by nitrogen deprivation, resulting in mating-incompetent pregametes, when cells are kept in the dark. For the completion of gametic differentiation light is required. Pregametes were treated with pharmacological compounds to influence the light-dependent conversion to mature gametes. Dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate, papaverine, and genistein were found to inhibit the progression of gametogenesis in the light. Treatment of pregametes in the dark with either staurosporine or papaverine resulted in their conversion to mature gametes. Apparently, papaverine has different effects in the dark and in the light; the effect of staurosporine suggested that a protein kinase C-like component inhibits the conversion of pregametes to gametes, a block that normally is relieved by illumination. This hypothesis was corroborated by the observation that activators of protein kinase C, N-heptyl-5-chloro-1-naphthalenesulfonamide, N- (6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, and the phorbolester phorbol-12-myristate 13-acetate inhibited gametogenesis in the light. Genistein and dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate were able to inhibit the dark activation caused by staurosporine treatment, suggesting that their targets work downstream from the "protein kinase C-like" kinase. Surprisingly, staurosporine and papaverine worked synergystically on the activation of pregametes in the dark.     

Chemotactic behavior of Chlamydomonas reinhardtii is altered during gametogenesis

Chemotactic behavior of Chlamydomonas reinhardtii is altered during the sexual life cycle. Unlike vegetative cells and noncompetent pregametes, mature gametes did not show chemotaxis to ammonium. Loss of chemotaxis to ammonium in mating-competent cells is controlled by gamete-specific genes that are common for both mating-type gametes. Change of chemotaxis mode requires the sequential action of the two environmental signals: removal of ammonium from the medium and light. The mutants lrg1, lrg3, and lrg4 affected in the light-dependent step of sexual differentiation exhibited the loss of chemotaxis to ammonium in the absence of light. These data indicate that there are common components in the signaling pathways that control change of chemotactic behavior and forming of mating competence in gametes.     

Chemotactic Behavior of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Is Altered During Gametogenesis

Chemotactic behavior of Chlamydomonas reinhardtii is altered during the sexual life cycle. Unlike vegetative cells and noncompetent pregametes, mature gametes did not show chemotaxis to ammonium. Loss of chemotaxis to ammonium in mating-competent cells is controlled by gamete-specific genes that are common for both mating-type gametes. Change of chemotaxis mode requires the sequential action of the two environmental signals: removal of ammonium from the medium and light. The mutants lrg1, lrg3, and lrg4 affected in the light-dependent step of sexual differentiation exhibited the loss of chemotaxis to ammonium in the absence of light. These data indicate that there are common components in the signaling pathways that control change of chemotactic behavior and forming of mating competence in gametes. Received: 14 May 2002 / Accepted: 21 June 2002     

Light dependence of sexual agglutinability in Chlamydomonas eugametos

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt ⁺ and mt ^- strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in chlamydomonas

In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.     

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)     

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     

Factors affecting the mating competence in the unicellular green algaChlamydomonas eugametos (Volvocales)

Routinely prepared gametes (by flooding 3 week-old agar cultures) showed about 80% mating competence if the opposite sexual partners were mixed together. The mating competence exhibited a strict dependence on the composition of the solution in which the cells were suspended before mixing; it decreased progressively with increasing concentration of nitrates. In contrast, no inhibiting effect was found if urea was used as the source of nitrogen. Other ions present in nutrient media did not show any effect. Mating activity varied according to the spectral composition of light, being higher with a blue light than with a red one. Blue light caused accumulation ofvis-à-vis pairs, which were blocked to form zygotes. Freshly released daughter cells in vegetatively grown synchronous cultures had a dual nature—vegetative and sexual one. In these daughter cells, similar rules were found for governing of mating competence to those valid for standard gametes obtained from flooded agar cultures. High mating competence was found in daughter cells released the during dark period in distilled water, nitrate-free media, in the presence of Mg²⁺ or Ca²⁺ ions, or in media containing urea. The conditions during which daughter cells are released and the conditions under which they mate can be considered crucial for expression of gametic nature as a mating competence.     

Action Spectrum for the Light-Dependent Step in Gametic Differentiation of Chlamydomonas reinhardtii

Differentiation of Chlamydomonas reinhardtii vegetative cells to gametes requires two environmental signals: nitrogen starvation and light. Vegetative cells incubated without nitrogen differentiate into pregametes. Pregametes can be converted into sexually mature gametes by irradiation with light. The action spectrum for the light-dependent step in gamete formation showed two maxima at 370 and 450 nanometers. This is similar to the spectrum of other blue light/ultraviolet light-A-absorbing photoreceptors.     

Flagellar adhesion between mating type plus and mating type minus gametes activates a flagellar protein-tyrosine kinase during fertilization in Chlamydomonas

When Chlamydomonas gametes of opposite mating type are mixed together, flagellar adhesion through sex-specific adhesion molecules triggers a transient elevation of intracellular cAMP, leading to gamete activation in preparation for cell-cell fusion and zygote formation. Here, we have identified a protein-tyrosine kinase (PTK) activity that is stimulated by flagellar adhesion. We determined that the protein-tyrosine kinase inhibitor genistein inhibited fertilization, and that fertilization was rescued by dibutyryl cAMP, indicating that the genistein-sensitive step was upstream of the increase in cAMP. Incubation with ATP of flagella isolated from non-adhering and adhering gametes followed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies showed that adhesion activated a flagellar PTK that phosphorylated a 105-kDa flagellar protein. Assays using an exogenous protein-tyrosine kinase substrate confirmed that the activated PTK could be detected only in flagella isolated from adhering gametes. Our results indicate that stimulation of the PTK is a very early event during fertilization. Activation of the PTK was blocked when gametes underwent flagellar adhesion in the presence of the protein kinase inhibitor staurosporine, but not in the presence of the cyclic nucleotide-dependent protein kinase inhibitor, H8, which (unlike staurosporine) does not block the increases in cAMP. In addition, incubation of gametes of a single mating type in dibutyryl cAMP failed to activate the PTK. Finally, flagella adhesion between plus and minus fla10-1 gametes, which have a temperature-sensitive lesion in the microtubule motor protein kinesin-II, failed to activate the PTK at elevated temperatures. Our results show that kinesin-II is essential for coupling flagellar adhesion to activation of a flagellar PTK and cAMP generation during fertilization in Chlamydomonas.     

Light Affects Flagellar Agglutinability in Chlamydomonas eugametos by Modification of the Agglutinin Molecules

The effect of light on the sexual competence of a light-sensitive mating type minus strain (mt⁻) of Chlamydomonas eugametos obtained by crossing a light-sensitive mating type plus strain (mt⁺) with a light-insensitive mt⁻ strain is described. As previously demonstrated for the mt⁺ parent, this study of one of the mt⁻ offspring shows that (a) a light-sensitive mechanism affects flagellar agglutinability in a rapid process that does not require protein synthesis; (b) only the activity of the flagellar agglutinins (glycoproteins responsible for agglutination) is susceptible to light while agglutinins on the cell body surface are not affected by light. We further demonstrate that (a) membrane vesicles naturally released from nonagglutinable dark gametes remain inactive. Extracts of these vesicles also remain inactive even though they contain agglutinin-like components; (b) inactive mt⁻ agglutinin is present in extracts of flagella from nonagglutinable dark gametes by comparison of its chromatographic, electrophoretic, and immunogenic properties with those of active agglutinin. When purified of all other flagellar proteins, it remains inactive; (c) a monoclonal antibody directed against the sexual agglutination site of the mt⁻ agglutintin discriminates between active and inactive agglutinins when present in a native state on the flagellar surface, but is unable to discriminate between them when they are denatured in sodium dodecyl sulfate-electrophoresis gels and blotted onto nitrocellulose. Taken collectively these observations suggest that light activation involves the chemical modification of the agglutinins in situ on the flagellar surface.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis inAmaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

Changes in photoreceptor currents and their sensitivity to the chemoeffector tryptone during gamete mating in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Chlamydomonas reinhardtii Dangeard generates photoreceptor currents (PCs) upon light excitation. These currents play a key role in the signal transduction chain for photomotility responses. We have previously found that inhibition of PCs by tryptone occurs only in gametes that display chemotaxis toward this agent, and is not observed in chemotactically insensitive vegetative cells. Here we show that the sensitivity to tryptone is characteristic of gametes of both mating types, and examine the influence of gamete mating on PCs and their sensitivity to tryptone. The amplitude of PCs increases after cell fusion, but the sensitivity of these currents to tryptone decreases upon flagellar adhesion and/or an increase in the intracellular cAMP concentration. Net chemotaxis toward tryptone is reduced in young zygotes compared to gametes. We conclude that gamete mating leads to rapid inactivation of a gamete-specific chemosensory system.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis in Amaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

The light induction of maize phosphoenolpyruvate carboxylase kinase translatable mRNA requires transcription but not translation

We have previously demonstrated that the level of translatable mRNA for phosphoenolpyruvate carboxylase kinase in maize leaves is increased in response to light ( Hartwell et al. 1996 ; Plant Journal 10 , 1071–1078). To identify the steps required for this increase, we have examined the effects of protein and RNA synthesis inhibitors. The RNA synthesis inhibitors actinomycin D and cordycepin (500 μ M ) strongly inhibited the light-induced increases in kinase translatable mRNA and the apparent phosphorylation state of phosphoenolpyruvate carboxylase, as judged by its sensitivity to inhibition by L -malate. The protein synthesis inhibitors cycloheximide and puromycin blocked the light-induced increase in the apparent phosphorylation state of phosphoenolpyruvate carboxylase but not the increase in kinase translatable mRNA. Indeed, the amount of phosphoenolpyruvate carboxylase kinase translatable mRNA after 3 h of illumination of leaves treated with either 1 m M puromycin or 100 μ M cycloheximide was double that in illuminated control leaves. Each inhibitor reduced the light-induction of two control genes, malic enzyme and pyruvate, phosphate dikinase. Thus the light induction of phosphoenolpyruvate carboxylase kinase translatable mRNA requires RNA synthesis, but not protein synthesis.     

Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii

When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db-cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide-dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.     

Phototropin plays a crucial role in controlling changes in chemotaxis during the initial phase of the sexual life cycle in<Emphasis Type="Italic"> Chlamydomonas</Emphasis>

During sexual differentiation, Chlamydomonas reinhardtii changes its chemotactic behavior in response to ammonium. Just like gamete formation, the change in chemotaxis mode is controlled by the sequential action of two environmental cues, removal of ammonium or nitrate from the medium and light. Thus, vegetative cells and mating incompetent pre-gametes, the latter being generated by nitrogen starvation in the dark, exhibit chemotaxis towards ammonium. Irradiation of pre-gametes results in a loss of chemotaxis and the gaining of mating competence. Incubation of these gametes in the dark resulted in their regaining chemotactic activity; re-illumination again resulted in its loss. Blue light was shown to be most effective in switching-off chemotaxis. RNA-interference strains with reduced levels of the blue-light receptor phototropin showed an attenuated inactivation of chemotaxis that could be partially compensated by the application of higher fluence rates, suggesting that these light responses are mediated by phototropin. The sharing of photoreceptor and signal transduction components as well as similar temporal patterns observed for changes in chemotaxis towards ammonium and gametic differentiation suggest an integration of the signaling pathways that control these two responses.Abbreviations mt Mating type - Phot Phototropin - RNAi RNA interference - TAP Tris–acetate–phosphate (medium) - TAP–N Nitrogen-free TAP (medium)