Journal of General Microbiology (1 987), 133, 1081-1088.
Printed in Great Britain
1081
Control of the Mating Competence Rhythm in Chlmydomonas eugametos
By R . D E M E T S , * A . M . T O M S O N , D. S T E G W E E A N D H . V A N D E N E N D E
Laboratorium voor Plantenfysiulogie, Universiteit van Amsterdam, Kruislaan 318,
1098 SM Amsterdam, The Netherlands
(Receioed I3 August I986 ;revised I9 December 1986)
Gametes of Chlamydomonaseugametus lose their mating competence during the light periods of
a 24 h light/dark (LD) cycle, and reacquire it in the dark periods. A diurnal rhythm in sexual
agglutinability of the flagella underlies these fluctuations. Under constant environmental
conditions the oscillation persists as an endogenous circadian rhythm. Under natural
environmental conditions the rhythm is cooperatively driven by endogenous and exogenous
signals. Exogenous control includes a decline in agglutinability upon illumination, which can be
countered by Diuron, and a rise in agglutinability upon lowering of the temperature. In LD,
pronounced fluctuations of the external pH are found that can be blocked by Diuron. The pH
rhythm does not interfere with the agglutinability rhythm. The control of other rhythms in
Chlamydumonas is discussed. As sexual agglutinability is due to well-characterized agglutinins
located on the flagellar membrane, this biological rhythm lends itself to further exploration at
the molecular level.
INTRODUCTION
Chlamydumunaseugametus is a unicellular oval-shaped green alga, propelled by two flagella. It
reproduces vegetatively by sporulation, whereby the cell divides into two, four or eight daughter
cells (Demets et al., 1985). Upon nutrient deficiency, a final division takes place to produce
gametes (Tomson et al., 1985). The gametes are morphologically identical to the vegetative
parents, but exhibit a specific mating reaction when confronted with gametes of the opposite
sex. The mating reaction comprises the following steps. As soon as partners recognize each
other, using their flagella as sensors, the contact is consolidated by agglutination. By
readjustments of the points of flagellar adhesion, the anterior ends of the cell bodies are brought
to close proximity. Then, the two gametes partially fuse by the formation of a common plasma
bridge, after which they resume swimming as a tandem organism, called a vis-a-vis pair. Several
hours later, the paired cells fuse completely into a zygote (Lewin, 1952; Mesland, 1976).
C . eugametus is isogamous, which means that the gametes of the two sexes are morphologically
identical. The sexes are referred to as mating type plus ( m t + )and mating type minus (mt-). Each
mating type possesses a specific set of agglutinins, located extrinsically on the flagellar
membrane of the gametes, which have been purified and characterized (Musgrave et al., 1981;
Homan et al., 1982; Klis et al., 1985). The agglutinins are large linear glycoproteins as shown by
electron micrographs (Klis et al., 1985; K. J . Crabbendam, personal communication). They play
a key role in the gamete-gamete contact.
Recently we reported regular daily fluctuations in mating competence of C . eugametos
(Tomson et al., 1985). Evidence presented in this paper shows that these variations are caused by
an endogenous rhythm in sexual agglutinability of the flagella.
Abbreviations: Diuron, 3-(3,4-dichlorophenyI)-l,l-dimethylurea;
LD, 12 h light/l2 h dark; LL, continuous
light; DD, continuous darkness.
0001-3651 R3 1987 SGM
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R . DEMETS A N D OTHERS
METHODS
Gametes.Chlamydomonaseugametos strains UTEX 9 (mt+)and UTEX 10 (mt-) from the Algal Collection at the
University of Texas at Austin, Texas, USA, were grown in Petri dishes on agar as described by Mesland (1976).
Gamete suspensions were obtained by flooding 2-weeksld cultures, grown under a 12 h light/l2 h dark regimen,
with distilled water.
Test conditions.All experiments were done at 19 "C unless stated otherwise. Illumination was provided by white
fluorescent tubes at 5500 lx (65 pE s-l m-2). The gamete suspensions were placed, according to the experimental
requirements, in a 12 h light/l2 h dark regimen (LD), in continuous light (LL) or in constant darkness (DD). All
experiments were repeated at least once, using different batches of gametes.
Quantification of Jagellar agglutinability. Gametes were fixed in glutaraldehyde (final concn 1.25%, v/v) for
10 min and washed twice in double-distilled water. The killed cells were mixed on a slide with live gametes of the
opposite mating type, giving an agglutination reaction that was observed under a light microscope. The reaction
was quantified by testing a binary dilution series of the fixed material. When the agglutinability of a sample of
fixed cells is referred to for instance as 2s, this means that up to the fifth twofold dilution step of this sample, a
positive agglutination reaction was scored when a constant quantity of living partner gametes was added. This
semi-quantitative bioassay has proved to produce reproducible results.
Mating competence test. To determine the percentage of sexually competent gametes in a given suspension, a
sample was mixed with gametes of the opposite mating type in a ratio of 1 :9, in triplicate, at 19 "C for 1 h, in the
light. Total cell density was always 1-2 x lo6 cells m1-I. The mixture was then fixed with glutaraldehyde (final
concn 1.25%, v/v) and counted for pairs (p) and single cells (s). The quotient lOp/2p+s gives the fraction of
competent gametes of the minor partner in the 1 :9 mixtures. Full details of the test are given by Tomson et al.
(1 986).
Inhibition ofphotosynthesis. A 0.1 ml volume of 1 mM-Diuron in ethanol was added to 10 ml gamete suspension.
This was sufficient to inhibit photosynthesis by at least 90%, as determined by the inhibition of O2 production,
using an oxygraph. The controls in these experiments also contained 1% (v/v) ethanol.
pH measurements. The external pH in gamete suspensions was continuously measured by a pH meter (Philips
PW 9409), coupled to a recorder. A multiple set-up of eight pH electrodes registered pH oscillations in test
suspensions and controls at the same time. The pH in each individual suspension was recorded every 16 min by an
automated system.
RESULTS
Circadian rhythm in flagellar agglutinability
Under LD conditions the mating competence of the gametes of both mating types of C.
eugametos shows a diurnal periodicity (Fig. 1 a). At the beginning of each light period, most cells
are able to form a pair with a gamete of the opposite mating type. During the course of the light
period, the percentage of competent cells steadily declines, often to zero. In the dark periods, the
2
3
4
1
2
3
Time (d)
Fig. 1. Mating competence rhythm of C. eugametos mt- in LD (a),DD (b) and LL (c),expressed as the
percentage of cells able to pair. Similar rhythms were found for mt+ gametes. (c) shows that the mating
competence disappeared in LL, but returned when the cells were placed in darkness again.
1
2
3
4
1
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Mating competence rhythm in Chlamydomonas
1083
100
h
80
E
Q)
60
40
12
24
Time (h)
xE
8
36
u)
and mating competence ( 0 )in C.
Fig. 2. Endogenous rhythms of flagellar agglutinability
eugumetos mt- in DD at 19 "C.t = 0 : start of the light period of the subjective LD regime. A, Boost in
agglutinability upon transfer of gametes from 19 "C to 9 "C at t = 4 h.
gametes regain their ability to pair. To explain these fluctuations, the assumption was made that
the competence was slowly suppressed by light. This idea originated from experiments on
gametogenesis that showed that only minor amounts of competent gametes were produced as
long as cultures were grown in LL (Tomson et al., 1985).If this hypothesis were valid, the mating
competence should have stayed permanently high when the cells were placed in continuous
darkness. In practice, the rhythm persisted in DD (Fig. 16). After a few cycles in DD, the
oscillation faded out, coinciding with the loss of vitality of the phototrophic cells, being deprived
of light. As all external conditions had been kept constant, this meant that the competence
rhythm originated from an endogenous oscillation. Since the period of the rhythm was close to
24 h, the oscillation must be considered as an endogenous, circadian rhythm (Biinning, 1960).
We previously established that in LD, the fluctuations in mating competence ran parallel to
fluctuations in sexual agglutinability (Tomson et al., 1985). Fig. 2 shows that in DD also, at a
constant temperature of 19 "C, flagellar agglutinability and mating competence remained
closely associated. As flagellar agglutinability is a prerequisite for successful mating, we
concluded that we were in fact looking at just one endogenous rhythm, that of flagellar
agglutinability.
Gametes were obtained from agar cultures that had been grown in LD. When the light and
dark periods of this LD regimen were reversed, a disturbed, non-rhythmic competence pattern
was found after one LD cycle, probably representing a transient stage of adjustment. After five
reversed LD cycles, gametes showed a phase-reversal in the competence rhythm, corresponding
to the newly imposed LD regime and running 12 h out of phase to the subjective LD regime (data
not shown). This entrainability by external synchronizers is a generally accepted property of
circadian rhythms (Edmunds, 1982).
Negative eflect of light
Comparison of Figs l(a) and l(6) shows that although the rhythm persisted in DD, the
oscillations were more pronounced in LD. This supports the initial hypothesis, that light has a
negative influence on mating competence. This idea was tested by placing gametes of high
competence in LL. The competence declined to an almost steady O x , indicating that there was
indeed an adverse effect of light (Fig. lc). Apparently in LD (Fig. la), the endogenously
generated decline in mating competence was reinforced by the light. Upon placing the cells back
in darkness, the competence returned to normal, showing that the prolonged illumination had
done no irreversible damage (Fig. 1c). The association between agglutinability and competence
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1084
R . DEMETS AND OTHERS
13;
I
I
Y
.-
I
12
24
36
48
Time (h)
'Fig. 3. External pH in gamete suspensions of C. eugametos. One batch of gametes was split into four
series at the end of a light period of the standard LD regime. (a) In LD; (b) in LD in the presence of
Diuron; (c) in LL; ( d ) in DD. The pH in the cell suspension was not affected by lophi-Diuron.
Table 1. Eflect of light and Diuron on flagellar agglutinability and mating competence in
C . eugametos
Gamete suspensions were kept in LD. After a standard 12 h light period, series (c) and (d) were
maintained in light and Diuron was added to series (b) and (d). Agglutinability and mating competence
were determined 12 h later.
(4
(b)
(4
(4
Illumination
Addition of
10 pM-Diuron
Agglutinability
titre
Mating
competence (%)
LD
LD
LL
LL
no
Yes
no
Yes
26
26
23
26
68
71
40
70
was also found in LL. Table 1 shows that the cells largely lost their agglutinability and
competence in LL, though the negative effect in this experiment was less dramatic than that
represented in Fig. 1(c). To investigate whether photosynthesis was involved in this light effect,
gametes were placed in LL in the presence of Diuron to block photosynthetic activity.
Agglutinability and competence were maintained at a significantly higher level than in the
controls (Table 1). Therefore, it was concluded that the negative effect of light was linked to
photosynthetic activity and that the Diuron treatment mimicked the effect of placing the cells in
the dark.
Rhythm in external pH
Upon illumination of gamete suspensions, a strong rise in the external pH was found, with a
decline to the original level in the dark periods. The rhythm was extremely pronounced: the
fluctations in the (non-buffered)gamete suspensions spanned more than four pH units (Fig. 3a).
The rhythm was completely blocked by Diuron (Fig. 3b), indicating that it was caused by
photosynthetic activity. No pH rhythm was found in LL or DD, where the pH was constant at
10.5 and 6.5 respectively (Fig. 3c, d ) . An attractive explanation for the photosynthesis-linked
loss of agglutinability in LL was the possibility that flagellar agglutinability was destroyed by the
excessively high external pH. However, when the mating process was tested over the pH range
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Mating competence rhythm in Chlamydomonas
1085
1
10
20
Temperature ("C)
30
Fig. 4. Effect of temperature on pair formation in C. eugumeros. A batch of gametes was split into five
equal portions that were mixed for 1 h at various temperatures with gametes of the opposite mating
type. No pre-incubation had taken place, so the gametes were equally agglutinable at the beginning of
the test.
Table 2. Combined efect of pre-incubation temperature and test temperature on pair formation in
C . eugametos
Pre-incubation started at the beginning of a dark period of the standard LD regime and lasted for 18 h.
The percentage of paired cells formed within 1 h upon mixing of the partners is given.
Pre-incubation temp. :
Test
\ - A - f
temp.
9°C
19 "C
19°C
9°C
92%
42%
43 %
6%
6-1 1 by resuspending gametes in various buffers (10 mM-Tris, HEPES, glycylglycine and
histidine) a normal agglutination reaction was seen at all pH values, in all buffers. Even when
the gametes had been pre-incubated for 12 h in a particular buffer, no inhibition of agglutination
was detected. Moreover, gametes lost their competence in LL when the medium was buffered at
pH 7.0 (data not shown). It was concluded that the loss of agglutinability induced by light is not
due to changes in external pH.
Temperature eflects
Temperature shifts can cause marked changes in endogenous rhythms, varying from phaseshifts to changes in the amplitude of the oscillation (Sweeney & Hastings, 1960). When gametes
were transferred from 19 "C to 9 "C in DD, an unexpected strong increase in flagellar
agglutinability was observed (Fig. 2). The fact that the work on Chfumydomonasgametes is done
at 19 "C arises from the general experience that the cells tend to lose their mating competence at
room temperature. Therefore, the effect of temperature on agglutinability may well span a wider
range than had previously been assumed. The lower the temperature, the more agglutinable the
cells seem to become. Fig. 2 shows that adaptation to the temperature drop took place rather
slowly, and was interwoven with the diurnal rhythmicity. In view of the increase in
agglutinability, it was expected that the sexual competence of the gametes would also be
enhanced at 9 "C. This was confirmed by the simple test shown in Table 2. A batch of gametes
was split into two; one half was kept overnight at 19"C, the other at 9"C, and mating
competence was then tested at 19 "C in both cases. The gametes that had previously been kept in
the cold formed about twice as many pairs (92%) as those that had been maintained at 19 "C
(43%). The higher overall agglutinability in the pre-cooled population resulted, as expected, in a
higher percentage of gametes being able to fuse with a partner. The experiment also revealed
that the enhanced mating competence that had gradually built up in the cold was not
immediately destroyed when the cells were re-exposed to 19 "C. This again suggests that the
agglutinability adjusts only slowly to new temperatures. The standard test temperature of 19 "C
was chosen in view of the fact that cell fusion is limited at lower temperatures (Weiss et ul., 1977;
Mesland & van den Ende, 1979; Fig. 4). This was also observed when gametes were preDownloaded from www.microbiologyresearch.org by
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R. DEMETS A N D OTHERS
incubated at 9 "C before fusion. Table 2 shows that in spite of the high agglutinability, pair
formation within 1 h at 9 "C (42%) was almost equal to that in the control at 19 "C (43%).
Obviously, at 9 "C the benefit of the increased agglutinability was nullified by the limited ability
to fuse.
DISCUSSION
A diurnal periodicity in mating competence, characterized by a maximum at the start of the
light period, has been found in numerous species within the Volvocales, including Astrophomene
gubernaculifera (Brooks, 1966), Chlamydomonas moewusii (W iese, 1984), Chlamydomonas
eugametos (Tomson et al., 1985; Wiese, 1984), Pandorina unicocca (Rayburn, 1974), Pandorina
morum (Coleman, 1979), Volvulina steinii (Carefoot, 1966) and Volvulina pringsheimii (Starr,
1962).As far as we know, there have been no reports on the control mechanism of these rhythms.
In C . eugametos, a circadian rhythm in sexual agglutinability of the flagella evidently
underlies the mating competence rhythm. Like most biological rhythms, the agglutinability
rhythm is driven endogenously in concert with exogenous influences (Aschoff, 1960). In DD, at
a constant temperature, the rhythm is solely endogenously controlled. In LD, two exogenous
effects interfere : (1) a suppressive effect of photosynthesis enhances the decline in
agglutinability ; (2) a temperature effect; since under non-laboratory conditions the light source
modulates the temperature, diurnal temperature changes presumably play an additional role in
the rhythm. Agglutinability rises upon lowering the temperature; this effect may cooperate with
the endogenous component of the rhythm that restores agglutinability at night under natural
day/night conditions.
Observations on various batches of gametes, of both mating types, suggested that the relative
contributions of the three effects (the endogenous oscillation, the light effect, the temperature
effect) may vary. Generally, all three effects were prominently present but sometimes one
of them was almost lacking. In exceptional cases, the entire diurnal rhythm was hardly
detectable.
Several diurnal rhythms have been discovered in Chlamydomonas. It is well known that the
vegetative division cycle can be phased in LD, resulting in the synchronous release of daughters
from the mother cells at every dark/light transition (Lloyd et al., 1982; Demets et al., 1985).
While Straley & Bruce (1979) have demonstrated that the division rhythm is endogenously
controlled, as it persists in DD (provided the cells are fed supplemental acetate), Spudich &
Sager (1980) have stated that the rhythm is brought about exogenously by direct (phasing) effects
of the alternating light and dark periods on the progress of the cells through the division cycle. It
seems more likely that each report covers a complementary part of the control mechanism and
that the cell division rhythm in LD is controlled by a combination of endogenous and exogenous
signals. A combined endogenous/exogenous control is very common in biological rhythms, but
the phenomenon is often overlooked (Hoffmann, 1976).
Other well-known diurnal rhythms in Chlamydomonasinclude photosynthesis and phototaxis.
Of course, both rhythms are, in LD, primarily exogenously controlled, for both are strictly lightdependent processes. Still, in continuous darkness (interrupted by short exposures to light)
persisting endogenous rhythms of photosynthetic capacity (Hoffmans-Hohn et al., 1984) and
phototaxis (Bruce, 1970) have been found.
In the cases of the cell division cycle, photosynthesis and phototaxis, the phasing of the
endogenous control corresponds to the phasing of the exogenous influences. The functional
significance of this co-phasing is evident: Chlamydomonas adapts to the LD cycles in order to
make optimal use of the offered light. To rationalize the mating competence rhythm is more
difficult. Although the endogenous control perfectly matches the exogenous influences, the
functional significance of the mating competence rhythm with respect to the sexuality is
obscure, as the daily decline of competence undoubtedly diminishes the rate of sexual
reproduction.
In LD, a marked rhythm in the external pH was detected. This pH rhythm was found in
vegetative cultures as well. Similar pH rhythms have been reported for other species of the
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Mating competence rhythm in Chlamydomonas
1087
Volvocales (Rayburn & Starr, 1974; Coleman, 1979; Hoffmans-Hohn et al., 1984). In C.
eugametos, this rhythm proved to be linked to photosynthetic activity. We have found no
indications of the involvement of nitrogen salts in this rhythm, as was suggested by Coleman
(1979) to explain the pH rhythm in P . morum. We suppose, instead, that the cells convert HCO?
into COz and OH- ions during the light periods, just as C. reinhardtii does (Tsuzuki, 1983). The
OH- ions are exported, causing the rise in pH, while the COz is retained for assimilation.
No clear-cut explanation for the rise in agglutinability upon a temperature drop has been
found, but the turnover of the agglutinins may play a role in this process. A slowed-down
degradation of the agglutinins in the cold perhaps leads to (temporary) accumulations of
agglutinins at the flagellar surface. Preliminary results indicate that after a shift-down in
temperature the agglutinability rhythm persists in DD without much alteration in the period. A
temperature-compensated period length is a common feature in many endogenous rhythms
(Sweeney & Hastings, 1960).
Besides the slow decline in agglutinability upon prolonged illumination reported here, some
strains of C . eugumetos interestingly show an additional, rapid reaction to light (Kooijman et al.,
1987; Wiese, 1984). In these cases light is needed to activate the agglutinin molecules before
sexual agglutinability can be expressed. These strains are, consequently, sexually inactive in
DD. By interrupting the DD regime with short light intervals, enough to activate the cell
(15 min), we established that the endogenous rhythm is present in these strains as well. The
conclusion is that this light-dependent on/off switch must be envisaged as a final regulatory step,
that results in the expression of agglutinability at a level that is pre-set by the circadian rhythm.
The circadian rhythm of flagellar agglutinability in Chlamydomonas reported here may be a
useful, simple model system. In contrast to the cell-cycle-related phenomena (Straley & Bruce,
1979; Spudich & Sager, 1980; Donnan & John, 1983) the rhythm can be studied in one
generation of non-dividing individuals. Moreover, monoclonal antibodies have recently been
raised against the agglutinins and other components of the flagellar surface. This offers the
attractive possibility of probing this biological rhythm at the molecular level.
The authors would like to thank Dr H. F. Bienfait for the use of his excellent pH equipment.
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