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     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

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)     

Circadian rhythms of chemotaxis to ammonium and of methylammonium uptake in chlamydomonas

Chlamydomonas reinhardtii expresses a well-documented circadian rhythm of phototaxis, which peaks in the subjective daytime. We find that vegetative cells also express circadian rhythms of chemotaxis to ammonium and ammonium uptake (as gauged by uptake of [¹⁴C]methylammonium). The chemotaxis rhythm peaks in the subjective night. Methylammonium uptake is light dependent, and its rhythm peaks at subjective dawn. Unlike vegetative cells, gametes are not attracted to ammonium. We believe this to be the first report of a circadian rhythm of chemotaxis.     

Genes Involved in Light Control of Sexual Differentiation in Chlamydomonas Reinhardtii

Gamete formation requires the sequential action of two extrinsic cues, nitrogen deprivation and blue light. The mutants described here are specifically altered in the light-dependent step. Mutations lrg1, lrg3, and lrg4 overcome this light dependence while mutation lrg2 results in a delayed execution of the light-mediated step. The four mutations are linked. The recessive nature of the lrg1, lrg3, and lrg4 mutations implies that they encode elements of negative control in this light response pathway. Analyses of diploids suggest an interaction between the gene products of the mutated loci with a central role for lrg4. The lrg4 mutation is unique also because it overcomes the light dependence of Chlamydomonas zygote germination when present in homozygous form. These data indicate that there are common components in the signal chains that control gametogenesis and zygote germination.     

Phototaxis and chemotaxis of brown algal swarmers

Brown algae exhibit three patterns of sexual reproduction: isogamy, anisogamy, and oogamy. Unicellular swarmers including gametes and zoospores bear two heterogenous flagella, an anterior flagellum with mastigonemes (fine tripartite hairs) and a posterior one. In seawater, these flagellates usually receive physico-chemical signals for finding partners and good habitats. It is well known that brown algal swarmers change their swimming direction depending on blue light (phototaxis), and male gametes do so, based on the sex pheromones from female gametes (chemotaxis). In recent years, the comparative analysis of chemotaxis in isogamy, anisogamy, and oogamy has been conducted. In this paper, we focused on the phototaxis and chemotaxis of brown algal gametes comparing the current knowledge with our recent studies.     

Chemotaxis in the Green Flagellate Alga <Emphasis Type="Italic">Chlamydomonas</Emphasis>

Behavior of the green flagellate alga Chlamydomonas changes in response to a number of chemical stimuli. Specific sensitivity of the cells to different substances might appear only at certain stages of the life cycle. The heterogamous species C. allensworthii demonstrates chemotaxis of male gametes towards pheromones excreted by female gametes. In C. reinhardtii chemotaxis towards tryptone occurs only in gametes, whereas chemotaxis towards ammonium, on the contrary, only in vegetative cells. Chemotaxis to different chemical stimuli might involve different mechanisms of reception and signal transduction, elucidation of which has only recently begun. Indirect evidences show that the cells likely respond to tryptone with changes in the membrane electrical conductance. The recently completed project of sequencing the whole nuclear genome of C. reinhardtii provides the basis for future identification of molecular elements of the chemosensory cascade in this alga.__________Translated from Biokhimiya, Vol. 70, No. 7, 2005, pp. 869–879.Original Russian Text Copyright © 2005 by Govorunova, Sineshchekov.     

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.     

Analysis of gamete and zygote motility in Allomyces

To study the mechanisms of chemotaxis in eukaryotes, the motility patterns of the gametes and zygotes and the chemotactic responses of the male gametes of the lower eukaryote Allomyces macrogynus were examined. Dark-field microscopy of the male gametes showed a smooth swimming pattern interrupted by very brief ‘jerks’ of the cell body that caused a change in swimming direction. Female gametes had a slower swimming velocity than the males and underwent more jerks or turns which accounted for their sluggish motility. The zygotes swam with the fastest velocity and were observed to have a helical swimming pattern involving a continuous turning of the cell body, a behavior absent from the gametes. Introduction of female gametes that produce the chemoattractant sirenin brought about an immediate change in the behavior of the male gametes. They moved in spirals (or helices) towards the source of the chemoattractant (the female gametes), undergoing only a few jerks to reorient the male cells. When very near the female cells, or in high concentrations of added sirenin, many very short motility tracks were observed that finally resulted in contact between the two gamete types. The results indicate that the poor swimming ability of the female gametes facilitates gamete contact, resulting in as many as 30–40 male gametes clustered on a single female cell. Further, male gamete orientation to the sirenin gradient is caused by the chemoattractant suppressing the jerk motion.     

Gametic behavior in a marine green alga, Monostroma angicava: an effect of phototaxis on mating efficiency

The role of phototactic behavior of gametes was tested experimentally in the slightly anisogamous marine green alga Monostroma angicava Kjellman, and the effect of phototaxis on mating efficiency was discovered. Both male and female gametes showed positive phototaxis in response to a white light source. In contrast, they did not respond to a red light source. Their swimming velocity did not differ between these two illuminating light sources. It was, therefore, suggested that the search ability of the gamete itself might not vary between phototactic and non-phototactic conditions. The number of zygotes formed during the mating process may be expressed as the product of the number of encounters between male and female gametes and the fraction of encounters that result in sexual fusion. In this study, with high densities of male and female gametes mixed in test tubes, almost all minor (fewer in number) gametes fused sexually within 10 min. After dilution of the gamete suspensions by half, mating efficiency in test tubes illuminated by white light from above was higher than that in dark controls. This suggests that male and female gametes gathered at the water surface through their positive phototaxis, thus increasing the rate of encounters. Mating efficiency also decreased if the test tubes were illuminated from above by white light and also shaken. Since negative phototaxis is clearly shown in planozygotes, we suggest that positive phototaxis of male and female gametes in M. angicava is an adaptive trait for increasing the rate of gametic encounters rather than for the dispersal of zygotes as previously reported for zoospores of some marine algae. Received: 12 February 1999 / Revision accepted: 24 May 1999     

Phototactic responses of Chlamydomonas eugametos gametes before and after fusion

The phototactic behavior of Chlamydomonas eugametos gametes and vis-à-vis pairs was quantitated using a fully automated, computer-controlled microvideo image analysis system. Two different mt- (mating type minus) and one mt+ (mating type plus) strain, together with the two combinations of pairs were studied. One mt- strain of dark-adapted gametes was non-phototactic while the others were positively phototactic at all effective intensities of white light. The mt+ strain exhibited one of the strongest positive responses that has so far been reported in algae (r-values greater than 0.7). After sexual fusion, the mt+ cell powers the swimming vis-à-vis pair. Its phototactic behavior reversed on fusion, with the pairs swimming away from all effective light intensities, irrespective of whether its partner was formerly phototactic or not. However, when adapted to the dark for an hour or more, vis-à-vis pairs swam positively to the light. The ecological consequence could be that pairs settle and develop into zygotes under intermediate light intensities or at light-dark interfaces.     

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

Simulation of gamete behaviors and the evolution of anisogamy: reproductive strategies of marine green algae

In marine green algae, isogamous or slightly anisogamous species are taxonomically widespread. They produce positively phototactic gametes in both sexes. We developed a new numerical simulator of gamete behavior using C++ and pseudo-parallelization methods to elucidate potential advantages of phototaxis. Input parameters were set based on experimental data. Each gamete swimming in a virtual rectangular test tank was tracked and the distances between the centers of nearby male and female were measured at each step to detect collisions. Our results shed light on the roles of gamete behavior and the mechanisms of the evolution of anisogamy and more derived forms of sexual dimorphism. We demonstrated that not only gametes with positive phototaxis were favored over those without, particularly in shallow water. This was because they could search for potential mates on the 2-D water surface rather than randomly in three dimensions. Also, phototactic behavior clarified the difference between isogamy and slight anisogamy. Isogamous species produced more zygotes than slightly anisogamous ones only under the phototactic conditions. Our results suggested that sperm limitation might be easily resolved particularly in the slightly anisogamous species. Some more markedly anisogamous species produce the smaller male gametes without any phototactic devices and the larger positively phototactic female gametes. In such species, female gametes attract their partners using a sexual pheromone. This pheromonal attraction system might have played a key role in the evolution of anisogamy, because it could enable markedly anisogamous species achieve 2-D search efficiencies on the water surface. The mating systems appear to be tightly tuned o the environmental conditions of their habitats.     

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

夜光藻有性繁殖研究进展

夜光藻是全球最主要的赤潮生物之一,也是我国近海常见的浮游甲藻。根据营养方式分为异养的红色夜光藻和混合营养的绿色夜光藻,前者广泛分布于温带和亚热带近岸水域,后者仅分布于热带西太平洋、阿拉伯海、阿曼湾和红海。夜光藻的生活史包括无性繁殖和有性繁殖过程。少部分营养细胞自发转变为配子母细胞,启动了有性繁殖。每个配子母细胞可形成大量配子,具有横沟、纵沟和2根鞭毛,形态与裸甲藻接近。配子两两融合形成合子,合子不经过休眠孢囊阶段直接发育成新的营养细胞。目前,对配子母细胞形成的调控机制、合子发育的影响因素的认识还存在分歧。研究发现,营养细胞经过一定次数的二分裂后都会转变为配子母细胞,而配子的存在能够中止此过程,使营养细胞继续进行二分裂。因此,有性繁殖可能通过产生新个体对种群增长做出贡献,还可能通过释放配子维持无性繁殖,进而促进种群增长。配子在相模湾水域全年都有分布,其丰度峰值与营养细胞丰度峰值同步或提前出现,配子的大量出现可能是赤潮形成的必要条件。对有性繁殖的研究佐证了夜光藻在甲藻的系统进化中处于较为古老的地位。此外,还简单介绍了研究夜光藻有性繁殖的主要方法,回顾了国内的夜光藻研究,并对相关研究进行了展望。     

Photoresponses of wild-type and mutant dikaryons ofChlamydomonas

Photoaccumulation and random motility of wild-type and mutant gametes and dikaryons ofChlamydomonas reinhardtii were evaluated with quantitative assays and compared with those of vegetative cells. Gametes exhibited behavior similar to that of vegetative cells. Dikaryons constructed from (+) and (−) wild-type gametes exhibited strong photoaccumulation in the presence of a stimulus and normal random swimming in red light, which shows that the activity of flagella and other components from two cells can be integrated and coordinated to permit appropriate behavior. Dikaryons from crosses of the wild type with mutants exhibited intermediate photoaccumulation. suggesting that neither phenotype is dominant. In contrast, crosses between an abnormally swimming mutant and normally motile strains showed that wild-type swimming was dominant. Partial complementation of mutant photoresponse phenotypes occurred in some crosses, but recovery of fully normal behavior was not observed.     

On the evolution of anisogamy from isogamous monoecy and on the origin of sex

A new hypothesis is established for the evolution of anisogamy in isogamous monoecious haploid unicells. Contrary to present concepts, it is assumed that in such isogamous ancestors the genetically identical gametes are bipolarly different as (+) and (?). The gametic differentiation of a monoecious taxon may be perceived as a phenocopy of that of a related dioecious form. The two gametic phenotypes result from alternative pathways of sexual differentiation. The alternative must be controlled by a sensible switch mechanism that responds to minor differences between vegetative cells when gametogenesis is triggered. Cell-size dependent factors are assumed to be able to influence the switch mechanism causing bigger cells to be of one sex, smaller cells of the other. Fertilization then occurs selectively between big and small gametes because of their sexual difference. Size-different gametes within one species may arise from different types of gametogenesis depending on how many gametes one vegetative cell produces. These assumptions, i.e. the bipolarity of isogametes, in monoecious taxa, size-dependent sex determination, and different types of gametogenesis within one taxon, are justifiable since they are realized independently from each other in various species. It is postulated that mutational fixation of the size-dependent sex determination in such monoecious species creates cell lineages producing sex-different macro- and microgametes and establishes constitutional anisogamous dioecy. The proposed hypothesis clearly separates the evolution of anisogamy as a problem of sexual reproduction from the evolution of sex per se, sex being defined as a bipolarity effecting fertilization.     

Chemotaxis of Caenorhabditis elegans during simultaneous presentation of two water-soluble attractants, l-lysine and chloride ions

Lysine and chloride ions are water-soluble attractants for Caenorhabditis elegans. When chemotaxis behavior to either of these attractants was assayed separately, the radial concentration gradients of 3 M lysine and 0.1 M ammonium chloride had similar potencies for attracting worms. However, when the concentration gradients of lysine and ammonium chloride at these concentrations were presented simultaneously, worms preferred lysine to ammonium chloride more than expected from the results obtained in separate experiments, suggesting the presence of an interaction between these two sensory information pathways within the nervous system. Chemotaxis behavior toward the radial concentration gradient of one of these attractants superimposed on a uniform concentration of the other attractant showed that the chemotaxis was augmented or attenuated by the ammonium chloride background depending on the background concentration, and attenuated by the lysine background, further supporting the interaction between the two sensory information pathways.     

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.