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Effects of nitrogen-containing compounds on the change in chemotaxis mode during gametogenesis of Chlamydomonas reinhardtii
Elena V. Ermilova, Zhanna M. Zalutskaya, Tatyana V. Lapina and Maksim M. Nikitin
Laboratory of Microbiology, Biological Research Institute of St. Petersburg State University, St. Petersburg, Russia
Summary
Chemotactic behavior of Chlamydomonas reinhardtii is changed during gametogenesis. Vegetative cells exhibit chemotaxis to ammonium ion. In the light, nitrogen deprivation induces the differentiation of vegetative cells into gametes that are not attracted to ammonium ion. Some L-amino acids affected the conversion of vegetative cells into chemotactically inactive cells. This strong effect of L-amino acids was due to their assimilation product, ammonium ion. Other utilizable sources of organic nitrogen, such as urea and adenine, were also able to block the loss of chemotaxis to ammonium. Intracellular ammonium ion generated by assimilation of organic nitrogen sources, e.g. amino acids, urea and purines, is supposed to repress the change in chemotaxis mode during gametogenesis of C. reinhardtii.
Key words: Chlamydomonas reinhardtii, chemotaxis, gametogenesis, nitrogen control
Introduction
The ability of unicellular organisms to differentiate in response to nutrient availability is essential to their survival in a changing environment (Lengeler et al., 2000). The biflagellate green alga Chlamydomonas reinhardtii represents one of the model protists of the study of mechanisms of sexual differentiation. Sexual reproduction in C. reinhardtii leads to formation of zygotes that, in contrast to vegetative cells, survive low temperatures and are not sensitive to desiccation (Harris, 1989).
The initial step in the sexual life cycle of C. reinhardtii is gametogenesis. During gametogenesis, C. reinhardtii vegetative cells that are normally haploid, are transformed into mating-competent gametes. Gametic differentiation of vegetative cells is induced by the depletion of an utilizable nitrogen source (usually ammonium ion) (Sager and Granick, 1954; Treier et al., 1989; Beck and Acker, 1992). Ammonium plays a crucial role as a preferred nitrogen source in C. reinhardtii (Fernández and Cárdenas, 1989; Fernández et al., 1998). Chlamydomonas has at least two ammonium ion carriers, the low-affinity ammonium
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transporter with a high maximum uptake velocity and the high-affinity ammonium transporter with a low maximum uptake velocity (Franco et al., 1987; Grossman and Takahashi, 2001). Alternative nitrogen sources such as amino acids, urea and purines can be assimilated by C. reinhardtii in the absence of ammonium (Fernández et al., 1998). It was shown that amino acids did not interfere with gametic differentiation (Beck and Haring, 1996). Ammonium ion itself, and not some metabolite, controls the formation of mating competent ability in cells (Matsuda et al., 1992). Mechanisms by which the cells are able to identify ammonium ion among other similar molecules remain unknown.
Gametic differentiation has been shown to be associated with changes not only in cell biochemistry and subcellular morphology (Beck and Haring, 1996) but also in chemotactic behavior of cells (Byrne et al., 1992; Ermilova et al., 2003). Unlike vegetative cells, gametes do not exhibit chemotaxis to ammonium. Recent studies have revealed that loss of chemotaxis to ammonium in mating-competent gametes is controlled by gamete-specific genes that are common for both mating-type gametes (Ermilova et al., 2003). Change in chemotaxis mode requires the action of two environmental signals: removal of ammonium from the medium and light (Ermilova et al., 2003). Here we report on the effects of various organic nitrogen-containing compounds on chemotactic activity of cells. Our results suggest that switch-off chemotaxis to ammonium in C. reinhardtii is regulated by intracellular level ofthis cation.
Material and methods
STRAINS AND CULTURE CONDITIONS
Chlamydomonas reinhardtii wild-type strain 620 (mt+), obtained from S. Purton (UCL, GB), was used as the tester strain. Cells were grown at 220C under continuous white light (8 W/m2) in a Tris-acetate-phosphate (TAP) medium (Gorman and Levin, 1965) or in an acetate-free TAP (TMP) medium, with ammonium or alternative nitrogen sources, such as Lamino acids (Sigma), urea (LKB) and adenine (Chemapol). These organic compounds were dissolved in distilled water and sterilized by filtration through membrane (0.2 ^m, Schleicher and Schuell, Germany). Growth was followed by measuring the cell number with the use of a counting chamber.
CHEMOTAXIS ASSAY
Cells were resuspended in nitrogen-free medium. Chemotactic responses were tested by counting the
number of cells that swam in darkness into rectangular capillaries (260 ^m x 450 ^m) filled with 3 ці of medium containing NH4NO3 (5 mM). This was compared with the number of cells entering capillaries filled with ammonium-free medium (Ermilova et al., 1993). Capillaries were closed at one end with Parafilm and the other end was submerged in the cell suspension for 10 min at 22°C. Chemotaxis index (CI) was calculated using the following equation:
number of cells entering capillaries filled with the test attractant
CI =----------------------------------------------------
number of cells entering capillaries filled with basic medium
Gametes were obtained by incubation of vegetative cells in nitrogen-free medium with continuous illumination (8 W/m2) for 24 h. Data are the means of triplicate determination from representative experiments.
2-OXOACID DETERMINATION
Cells grown in TAP-N medium supplemented with 2mM L-alanine were removed by centrifugation. Production of 2-oxoacid (pyruvic acid ) was determined by a modification of the method described (Borchers, 1977). To 0,5 ml of supernatant, which was contained pyruvic acid, was added 0.15 ml of 0.2 % (w/v) 2,4, dinitrophenylhydrazine in 2M HCl and after stirring, 0.5 ml of 2.5 M NaOH. After standing at room temperature for 10-15 min, to allow the color to develop, the absorbance at 445 nm was measured. Data are the means of triplicate determination from a representative experiment.
Results
EFFECT OF GLUTAMINE ON CHANGE IN CHEMOTAXIS MODE DURING GAMETOGENESIS
In the light, the depletion of ammonium ions induced the differentiation of vegetative cells into gametes that were unable to perform chemotaxis to ammonium (Fig. 1). Amino acids did not interfere with the formation of mating competence in gametes (Matsuda et al., 1992; Beck and Haring, 1996). To test whether the change in chemotaxis mode during gametogenesis may occur in the presence of amino acid, glutamine (final concentration 2 mM) was added to TMP-N medium at the start of gametogenesis. Figure 1 shows that glutamine did not prevent the formation of chemotactically inactive cells. However, addition of glutamine to cells resuspended in TAP-N medium resulted in a complete block of the alteration
б 0.5 J--------1------------1-----------1--------------1—
0 24 48 72
Time, h
Fig. 1. Effect ofL-glutamine on the loss ofchemotaxis to ammonium during gametogenesis. Vegetative cells were harvested and resuspended (0.1X106 cells/ml) in either NH4+-free medium (TMP-N (о) or TAP-N (о)) or in NH4+-free medium supplemented with glutamine (2 mM) (TMP-N (о ) or TAP-N (о)). Ammonium was removed at time 0.
in chemotactic activity to ammonium. The assimilation of extracellular glutamine by C. reinhardtii is strongly dependent on acetate supply (Muñoz-Blanco et al., 1990). We conclude that glutamine affected the conversion of vegetative cells into chemotactically inactive cells only during its assimilation.
CHEMOTAXIS TO AMMONIUM IS RETAINED UPON ADDITION
of L-amino acids, which are deaminated by cells
We have tested effects of various L-amino acids on switch-off chemotaxis to ammonium during gameto-genesis. Change in chemotactic activity of cells was detected in TAP-N medium supplemented with threonine, proline and glutamate (Table 1). C. reinhardtii cells did not grow on these amino acids (Fernández et al., 1998). Therefore, we next examined the effects of amino acids that are utilized effectively by C. reinhardtii for growth. Alanine, arginine, serine and phenylalanine prevented loss of chemotaxis in cells (Table 1). In contrast, alanine, serine and phenylalanine, like glutamine, had no effect on the formation of chemo-tactically inactive cells in TMP-N medium (data not shown). As reported previously (Piedras et al., 1992), C. reinhardtii cells are able to deaminate the L-amino acids extracellularly in a reaction mediated by a nonspecific L-amino-acid oxidase. This reaction required the presence of acetate in the medium. As a result, the corresponding 2-oxoacid, ammonium and hydrogen
peroxide are generated. The 2-oxoacids produced by deamination are not utilized (Muñoz-Blanco et al., 1990). For comparison, the kinetics for the accumulation of extracellular 2-oxoacid (pyruvic acid) and for the change in chemotactic activity are shown (Fig. 2). When pyruvic acid was accumulated in the medium, cells retained chemotaxis to ammonium. Accumulation of ammonium ion in the extracellular medium of TAP-amino-acid-growing cells was never detected (Muñoz-Blanco et al., 1990). Ammonium enters the cells rapidly (Fernández et al., 1998). These results suggest that ammonium provided by amino acids, and not amino acids per se, interfered with change in chemotaxis mode.
Arginine prevented the loss of chemotaxis even in the absence of acetate (Fig. 3). It was shown that arginine is the only amino acid which can be transported into the cells by a specific transport system induced by nitrogen starvation (Kirk and Kirk, 1978) Therefore, arginine can be assimilated by C. reinhardtii to yield intracellular ammonium. We propose that not extracellular but intracellular ammonium blocks the alteration in chemotaxis mode.
EFFECTS OF UREA AND ADENINE ON THE CHANGES IN CHEMOTAXIS MODE
Urea is incorporated into C. reinhardtii cells by an active transport mechanism (Williams and Hodson, 1977). In cells, urea is hydrolyzed to ammonia by the enzyme complex ATP:urea amidolyase (Leftley and Syrett, 1973). Addition of urea to the nitrogen-free medium completely prevented the loss of chemotaxis to ammonium (Fig. 4, A). Our results suggest that urea may serve as a source of ammonium ions to block
Table 1. Effects of L-amino acids on the loss of chemotaxis to ammonium during gametogenesis
Amino acid Cell growth Chemotaxis to ammonium
None - -
Threonine - -
Proline - -
Glutamate
Alanine + +
Arginine + +
Serine + +
Phenylalanine + +
Notes: vegetative cells were resuspended in TAP-N medium supplemented with various amino acids. After 24, 48 and 72 h of incubation, cell number and chemotaxis to ammonium were determined.
0 6 24 48
Time, h
Fig. 2. Kinetics of accumulation of 2-oxoacid and the change in chemotactic activity cells to ammonium. Vegetative cells were transferred to TMP-N medium or TMP-N medium supplemented with 2mM of L-alanine (TMP-N (- -) or TAP-N (—)). At the times indicated, the extracellular concentration of pyruvic acid (•) and chemotaxis index (a) were determined as described in Materials and Methods.
switch-off chemotaxis to ammonium. These data were supported by the experiment with adenine that also affected the change in chemotactic activity of cells to ammonium (Fig. 4, B). Purines can be assimilated phototrophically by C. reinhardtii to yield intracellular urea and then ammonium (Pineda and Cárdenas, 1996). The present results demonstrate that the intracellular ammonium ion may be a repressor of change in chemotaxis mode during gametogenesis.
Discussion
Gametes differ from vegetative cells by their mating ability. In the light, the removal of ammonium from
24 48 72
Time, h
Fig. 3. Effect of L-arginine on chemotaxis mode of cells. Vegetative cells were harvested and resuspended (0.1x106 cellc/ml) in either NH4+-free medium (TMP-N $) or TAP-N p)) or in NH4+-free medium supplemented with arginine (2mM) (TMP-N (a) or TAP-N (a)). Ammonium was removed at time 0.
culture medium of C. reinhardtii not only results in the formation of mating competence but also in switch-off chemotaxis to ammonium in gametes (Fig. 1). Effects of ammonium on the cell differentiation have been described in other organisms, e.g. Saccharomyces cerevisiae (Lengeler et al., 2000), Schizosaccharomyces pombe (Egel et al., 1990; Nielsen, 1993) and Dictyo-stelium discoideum (Wang and Schaap, 1989).
Amino acids may be used as sole nitrogen sources by C. reinhardtii (Vallon et al., 1993). As was shown, amino acids may influence the switch-off chemotaxis to ammonium (Table 1, Figs 1-3). One possible reason
0 24 48 72 96 0 24 48 72
Time, h Time, h
Fig. 4. Effects ofurea (A) and adenine (B) on cell growth and chemotactic activity of cells to ammonium. Vegetative cells were transferred to TMP-N medium or TMP-N medium supplemented with urea (200 ^g/ml) or adenine (500 ^g/ml). At the times indicated, cell number (•) and chemotaxis index (a) were determined. The broken lines represent cell number and chemotaxis index observed when cells were incubated in TMP-N medium.
Fig. 5. Proposed pathways for the production of ammonium ion that control change in chemotaxis mode of C. reinhardtii. The depletion of ammonium from the medium triggers change in chemotactic activity to this cation. A supply of ammonium ion blocks switch-off chemotaxis (T-bar). AAO- L-amino-acid oxidase.
for this result was that ammonium which is produced by L-amino-acid oxidase completely blocked the loss of chemotaxis in the differentiating cells. Urea and adenine also interfered with change in chemotaxis mode (Fig. 4). Urea and purines catabolized to yield ultimately ammonium (Fernández et al., 1998). We assume that the change in chemotaxis mode is triggered by a decrease in concentration of intracellular ammonium. According to our model, an intracellular threshold level of this cation may be increased by the assimilation of alternative nitrogen sources, e.g. amino acids, urea and purines, and this increase in concentration represses switch-off chemotaxis (Fig. 5). The previous study showed that cultures grown on amino acids were gametogenic (Matsuda et al., 1992). It was suggested that the program of differentiation of mating-competent cells is initiated before ammonium becomes available from the deamination of amino acid (Beck
and Haring, 1996). At the same time, this ammonium prevents the program of changes in chemotaxis mode (Table 1, Figs 1-3). We therefore propose that the expression of genes responsible for switch-off chemotaxis to ammonium may take place at the late phase of gametogenesis. As a result, ammonium generated by utilization of alternative nitrogen sources may block gene systems expressed later in gametogenesis. The mode by which ammonium deprivation is sensed and converted into a biological signal is still unknown.
Acknowledgements
This work was supported by a grant from RFBR № 04-00-48844.
References
Beck C.F. and Acker A. 1992. Gametic differentiation of Chlamydomonas reinhardtii. Plant Physiol. 98, 822-826.
Beck C. and Haring M. 1996. Gametic differentiation of Chlamydomonas. Int. Rev. Cytol. 168, 259-302.
Borchers R. 1977. Allantoin determination. Anal. Biochem. 79, 612-613.
Byrne T.E., Wells M.R. and Johnson C.H. 1992. Circadian rhythms of chemotaxis and of methylammonium uptake in Chlamydomonas reinhardtii. Plant Physiol. 98, 879-886.
Ermilova E.V, Zalutskaya Zh.M. and Gromov B.V. 1993. Chemotaxis towards sugars in Chlamydomonas reinhardti. Curr. Microbiol. 27, 47-50.
Ermilova E.V., Zalutskaya Zh.M., Lapina T.V. and Nikitin M.M. 2003. Chemotactic behavior of Chlamy-domonas reinhardtii is altered during gametogenesis. Curr. Microbiol. (in press).
Egel R., Nielsen O. and Weilguny D. 1990. Sexual differentiation in fission yeast. Trends Genet. 6, 369-373.
Fernández E. and Cárdenas J. 1989. Genetics and regulatory aspects of nitrate assimilation in algae. In: Molecular and genetics aspects of nitrate assimilation (Eds. Wray J.L. and Kinghorn S.). pp. 101-124.
Fernández A., Galván A. and Quesada A. 1998. Nitrogen assimilation and its regulation. In: The molecular biology of chloroplasts and mitochondria in Chlamydomonas (Eds. Rochaix J.-D., Goldschmidt-Clermont M. and Merchant S). Kluwer, Dordrecht. pp. 637-659.
Franco A.R., Cárdenas J. and Fernández E. 1987. A mutant of Chlamydomonas reinhardtii altered in the transport of ammonium and methylammonium. Mol. Gen. Genet. 206, 414-418.
Gorman D.S. and Levin R.P 1965. Cytochrome and plastocyanin: their sequences in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA. 54, 1665-1669.
Grossman A. and Takahashi H. 2001. Macronutrient utilization by photosynthetic eukaryotes and the fabric of interactions. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 163-210.
Harris E. 1989. The Chlamydomonas sourcebook: A comprehensive guide to biology and laboratory use. Acad. Press, San Diego.
Kirk D.L. and Kirk M.M. 1978. Carrier-mediated uptake of arginine and urea by Chlamydomonas reinhardtii. Plant Physiol. 61, 556-560.
Leftley J.W. and Syrett P.J. 1973. Urease and ATP:amidolyase activity in unicellular algae. J. Gen. Microbiol. 77, 109-115.
Lengeler K.B., Davidson R.C., D’Souza C., Toshiaki H., Shen W.-C., Wang P., Pan X., Waugh M. and Heitman J. 2000. Signal transduction cascades regulating fungal development and virulence. Microbiol. Mol. Biol. Rev. 64, 746-785.
Muños-Blanco J., Hidalgo-Martinez J. and Cárdenas J. 1990. Extracellular deamination of Lamino acids by Chlamydomonas reinhardtii cells. Planta. 182, 194-198.
Matsuda Y., Shimada T. and Sakamoto Y. 1992. Ammonium ions control gametic differentiation and dedifferentiation in Chlamydomonas reinhardtii. Plant Cell Physiol. 33, 909-914.
Nielsen O. 1993. Signal transduction during mating and meiosis in S. pombe. Trends Cell Biol. 3, 60-65.
Piedras P, Pineda M., Muños-Blanco J. and Cárdenas J. 1992. Purification and characterization of an L-amino acids oxidase from Chlamydomonas reinhardtii cells. Planta. 188, 13-18.
Pineda M. and Cárdenas J. 1996. Transport and assimilation of purines in Chlamydomonas reinhardtii. Sci. Mar. 60, 195-201.
Sager R. and Granick S. 1954. Nutritional control of sexuality in Chlamydomonas reinhardtii. J. Gen. Physiol. 37, 729-742.
Treier U., Fuchs S., Weber M., Wakarchuk W.W. and Beck C. 1989. Gametic differentiation in Chlamydomonas reinhardtii: light dependence and gene expression patterns. Arch. Microbiol. 152, 572-577.
Vallon O., Bulté L., Kuras R., Olive J. and Wollman F.A. 1993. Extensive accumulation of an extracellular L-amino acid oxydase during gameto-genesis of Chlamydomonas reinhardtii. Eur. J. Biochem. 215, 351-360.
Wang M. and Schaap P. 1989. Ammonia depletion and DIF trigger stalk cell differentiation in intact Dictyostelium discoideum slugs. Development. 105, 569-574.
Williams S.K.I. and Hodson R.C. 1977. Transport of urea at low concentrations in Chlamydomonas reinhardtii. J. Bacteriol. 130, 266-273.
Address for correspondence: Elena V. Ermilova. Laboratory of Microbiology, Biological Research Institute of St. Petersburg State University, Oranienbaumskoye sch. 2, Stary Peterhof, St.Petersburg, 198504. Russia. E-mail: [email protected]
The manuscript is presented by A.V.Goodkov
