Plant Cell Physiol. 44(10): 1081–1087 (2003)
JSPP © 2003
Mucilage Secretion Regulated by Sex Pheromones in Closterium peracerosumstrigosum-littorale Complex
Satoko Akatsuka 1, 5, Hiroyuki Sekimoto 2, 6, Hiroaki Iwai 3, Ryo-hei Fukumoto 4 and Tadashi Fujii 1
1
Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193 Japan
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902 Japan
3
Institute of Biological Sciences, University of Tsukuba, Ibaraki, 305-8572 Japan
4
Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193 Japan
2
;
When mating-type plus (mt+) and minus (mt–) cells of
the Closterium peracerosum-strigosum-littorale complex
were mixed in nitrogen-depleted mating medium, secretion
of mucilage containing uronic acid from cells was markedly activated and the mucilage accumulated around the
cells. Substances with the ability to stimulate mucilage
secretion from mt+ and mt– cells were detected in media in
which mt– and mt+ cells had been separately cultured,
respectively. We designated the active substances secreted
from mt+ and mt– cells mucilage secretion-stimulating pheromone (MS-SP)-plus and MS-SP-minus, respectively. Activity of MS-SP-plus and MS-SP-minus decreased to 20%
level by incubation at 80°C for 10 min. Light was indispensable for the secretion of mucilage. The secretion of MS-SPplus and MS-SP-minus decreased with dark treatment.
MS-SP-plus eluted at around 95 k from a gel filtration column, and reacted with antibodies against two subunits of
protoplast-release-inducing protein (PR-IP), which induces
protoplast release from mt– cells. MS-SP-minus eluted at
around 20 k from a gel filtration column, and reacted with
an antibody against the PR-IP inducer, which induces the
secretion of PR-IP from mt+ cells. In addition, purified PRIP and PR-IP inducer stimulated mucilage secretion from
mt– and mt+ cells, respectively. These results strongly suggested that MS-SP-plus and MS-SP-minus were the same
molecules as the PR-IP and the PR-IP inducer, respectively.
Keywords: Closterium — Mucilage — Sex pheromone —
Sexual reproduction.
Abbreviations: MS-SP, mucilage secretion-stimulating pheromone; mt+, mating-type plus; mt–, mating-type minus; PR-IP, protoplastrelease-inducing protein; SCD, sexual cell division; SCD-IP, sexual
cell division inducing pheromone.
1971). Its sexual reproductive process consists of several
sequential steps: sexual cell division (SCD) which is responsible for the differentiation from vegetative cells into sexually
competent cells (gametangial cells), pair formation of sexually
competent cells, formation of conjugation papillae, release of
protoplasts from the cells, and fusion of protoplasts to form a
zygote (Ichimura 1971).
It is well known that sex pheromones influence sexual
reproduction (Ichimura 1971, Hogetsu and Yokoyama 1979,
Coesel and de Jong 1986, Kato et al. 1981, Fukumoto et al.
1998, Sekimoto 2000). In particular, protoplast-releaseinducing protein (PR-IP) and its inducing pheromone (PR-IP
inducer) have been purified from the Closterium peracerosumstrigosum-littorale complex (Sekimoto et al. 1990, Nojiri et al.
1995), and cDNAs encoding these pheromones have been
cloned (Sekimoto et al. 1994a, Sekimoto et al. 1994b,
Sekimoto et al. 1994c, Sekimoto et al. 1998). Another protein,
SCD-inducing pheromone (SCD-IP), has been detected and
purified from Closterium ehrenbergii (Fukumoto et al. 1997,
Fukumoto et al. 2002).
To effectively promote the activities of these pheromones
in the sexual reproduction process, it is indispensable that sexually competent cells be in relatively close proximity. It is
known that Closterium exhibits a gliding locomotory behavior
(Domozych et al. 1993) and that the forceful extrusion of mucilage from one pole of the cell causes the cell to glide in the
opposite direction. It is also believed that in some desmids the
release of mucilage by a cell facilitates adhesion of the cell to a
solid object (Surek and von Sengbusch 1981). Despite several
reported cytological and biochemical analyses of mucilage
from Closterium, little is known about the role of mucilage
secretion in its sexual reproduction.
In this report, we describe mucilage secretion regulated by
sex pheromones in the sexual reproduction process of the C.
peracerosum-strigosum-littorale complex.
Introduction
Results
Closterium is a genus of unicellular charophycean algae
with crescent- or spindle-shaped cells (Pickett-Heaps and Fowke
Secretion of mucilage containing uronic acid from cells
was detected after visualization with alcian blue staining (Fig.
5
6
Present address: Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho,
Koganei, Tokyo, 184-8588 Japan.
Corresponding author: E-mail, [email protected]; Fax, +81-3-5454-6644.
1081
1082
Mucilage secretion by Closterium pheromones
Fig. 1 Photographs of Closterium cells and secreted uronic acids stained with alcian blue dye. (A–C) Features of mucilage secretion from cell
cultures. mt+ (A) and mt– cells (B) were cultured separately or mixed (C) in MI medium for 48 h. Scale bar = 500 mm. (D–H) Magnified photographs of cells undergoing the sexual reproduction process. (D) Cell during sexual cell division, (E) cells after sexual cell division, (F) cells forming papillae, (G) protoplast, (H) zygote. Scale bar = 20 mm.
1A–C). The mucilage was secreted from both mt+ and mt– cells
cultured separately in nitrogen-depleted mating medium (MI
medium; Ichimura 1971). Much greater amounts of mucilage
were secreted from mt– cells than from mt+ cells. When mt+
and mt– cells were cultured as a mixture in MI medium, the
amount of mucilage secreted increased markedly and a large
amount of mucilage accumulated, surrounding the cells. A
magnified view of the cells revealed that mucilage secretion
occurred at the division face during SCD, and that the mucilage was distributed in the shape of a cord, connecting the
dividing cells and surrounding papillae, protoplasts and zygotes
(Fig. 1D–H).
To evaluate quantitatively the mucilage secretion, both mt+
and mt– cells were cultured either separately or mixed together
in MI medium, and the amounts of total carbohydrate and
uronic acid secreted into culture medium were measured.
When the mt+ and mt– cells were cultured together, the secretion of both carbohydrate and uronic acid increased remarkably compared to separate cultures (Fig. 2). These results suggested that mucilage secretion by Closterium was influenced
by mutual communication between mt+ and mt– cells. The
results of glycosyl composition analysis indicated that the
sugar compositions were almost identical in all cases (data not
shown).
mt+ and mt– cells were separately cultured in conditioned
media in which mt– and mt+ cells had been previously cultured
in MI medium under continuous light. After various intervals,
the amount of uronic acid was determined (Fig. 3). Stimulation
of mucilage secretion was observed in the conditioned media.
The active substances that stimulated mucilage secretion, originating from mt+ and mt– cell cultures, were designated MS-SPplus and MS-SP-minus, respectively.
When mt+ and mt– cells were separately cultured in darkness, the amounts of secreted mucilage were remarkably decreased, even in respective conditioned media in which mt– and
mt+ cells had been cultured separately in the light (Table 1, 2).
Light was also indispensable for the secretion of both MS-SPminus and MS-SP-plus, although a lower level of stimulating
effect was observed in conditioned medium from mt+ cells
incubated in darkness (Table 2). Activity of MS-SP-plus and
MS-SP-minus decreased to 20% of the previous level by incubation at 80°C for 10 min (Fig. 4).
Mucilage secretion by Closterium pheromones
1083
Fig. 2 Time course of mucilage secretion. mt+ and mt– cells were cultured separately or together under continuous light. At various time points,
amounts of total carbohydrate (A) and uronic acid (B) were determined for the secreted mucilage from mt+ cells (square), mt– cells (triangle), and
mixture of mt+ and mt– cells (circle). Error bars indicate standard errors.
Fig. 3 Effects of conditioned media on mucilage secretion from Closterium. mt+ (A) and mt– (B) cells were separately cultured in conditioned
media (circle), in which the cells of opposite mating type had been precultured for 72 h in the light (see Materials and Methods), or in MI medium
(triangle). After various durations, the amount of uronic acid was determined. Error bars indicate standard errors.
Conditioned media, in which mt+ and mt– cells had separately been cultured for 48 h, were applied directly to Sephacryl
S-300 HR and Sephacryl S-100 HR columns, respectively. MSSP-plus eluted at around 95 k on the former column, and
reacted with antibodies against the 42- and 19-kDa subunits of
the PR-IP, which induces protoplast release from mt– cells
(Sekimoto et al. 1990) (Fig. 5). MS-SP-minus eluted at around
20 k on the latter column, and reacted with an antibody against
1084
Mucilage secretion by Closterium pheromones
Table 1 Effects of dark treatment on each stage of mucilage
secretion from mt+ cells
Conditions for
mucilage secretion
Light
Dark
Light
Dark
Light
Dark
Medium
Mucilage (mg uronic
acid / 106 cells)
MI
MI
conditioned (L)
conditioned (L)
conditioned (D)
conditioned (D)
5.41
ND
30.87
3.61
2.41
ND
mt+ cells were cultured under light or darkness, in which mt– cells had
been cultured under light [conditioned (L)] or darkness [conditioned
(D)]. As a control, MI medium was also used instead of the conditioned
media. The amount of uronic acid was determined. ND, not detected.
Table 2 Effects of dark treatment on each stage of mucilage
secretion from mt– cells
Conditions for
mucilage secretion
Light
Dark
Light
Dark
Light
Dark
Medium
Mucilage (mg uronic
acid / 106 cells)
MI
MI
conditioned (L)
conditioned (L)
conditioned (D)
conditioned (D)
26.32
ND
118.34
7.86
67.16
5.65
mt– cells were cultured under light or darkness, in which mt+ cells had
been cultured under light [conditioned (L)] or darkness [conditioned
(D)]. As a control, MI medium was also used instead of the conditioned
media. The amount of uronic acid was determined. ND, not detected.
the PR-IP inducer, which induces the secretion of PR-IP from
mt+ cells (Nojiri et al. 1995) (Fig. 6). Meanwhile, the PR-IP
and PR-IP inducers were purified and applied to cultures of mt–
and mt+ cells in MI medium, respectively. Stimulation of mucilage secretion was observed in both cases (Table 3).
Discussion
The secretion of mucilage containing uronic acid from the
C. peracerosum-strigosum-littorale complex occurs not only in
the sexual reproductive process, but also in the vegetative stage.
Closterium cells tended to form aggregates in resting cultures
during vegetative reproduction. It has been suggested that mucilage has a role in promoting aggregation of cells (Domozych et
al. 1993).
Mucilage accumulated at the division face during cell
division, and was distributed in the form of a cord connecting
two dividing cells. During sexual reproduction, mucilage surrounds papillae, protoplasts and zygotes (Fig. 1), possibly as a
protective structure. These results strongly suggest that accumulation of mucilage facilitates cell adhesion and cell-to-cell
communication.
Fig. 4 Effects of heat treatment on the mucilage secretion-stimulating
activities. The media containing MS-SP-plus (closed circle) and MSSP-minus (open circle) were exposed to various temperatures for
10 min and then assayed for mucilage secretion-stimulating activities.
As shown in Fig. 2 and 3, the secretion of mucilage was
markedly stimulated by the action of MS-SP-plus and MS-SPminus. Both MS-SPs were heat-labile (Fig. 4) and were not
secreted from cells under dark conditions (Table 1, 2). These
characteristics were the same as those of PR-IP (Sekimoto et
al. 1990) and PR-IP inducer (Sekimoto et al. 1993). These
results are also in full agreement with the fact that light is necessary for sexual reproduction in Closterium (Ichimura 1971,
Sekimoto 2000). In addition, the results shown in Fig. 5, 6 and
Table 3 strongly suggest that PR-IP and PR-IP inducer have
activities for stimulation of mucilage secretion from mt– and
mt+ cells as MS-SP-plus and MS-SP-minus, respectively.
Recently, two SCD-IPs, which are responsible for SCD of
respective mating-type cells, were detected in the mating culture medium of C. peracerosum-strigosum-littorale complex
(Tsuchikane et al. 2003). One (SCD-IP-minus) was released
from mt– cells in the light, was heat-labile and had an apparent
molecular mass of 20 k. The other (SCD-IP-plus) was released
from mt+ cells in the light, was heat-labile and had an apparent
molecular mass of 95 k. These physiological or biochemical
characters are also similar to those of PR-IP inducer and PR-IP.
In addition, a cDNA encoding SCD-IP from C. ehrenbergii was
cloned and the deduced amino acid sequence showed high level
of similarity with that of PR-IP inducer (Fukumoto et al. 2003).
From these results, it is possible to consider that PR-IP (MSSP-plus) and PR-IP inducer (MS-SP-minus) also have activities for SCD-IP-plus and SCD-IP-minus, respectively. For the
progress of sexual reproduction, respective biological activities
of pheromones must be critically regulated through these proc-
Mucilage secretion by Closterium pheromones
1085
Fig. 5 Gel filtration of MS-SP-plus activity on a Sephacryl S-300 HR
column. Conditioned medium, in which mt+ cells had been cultured
was lyophilized and applied to a Sephacryl S-300 HR column and
eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM
NaCl, at a flow rate of 5 ml h–1. Fractions of 2 ml were collected and
assayed for mucilage secretion-stimulating activity. Broken line indicates control secretion level in MI medium. Arrows indicate the elution points of marker proteins (kDa): thyroglobulin (669), catalase
(232), ovalbumin (45), ribonuclease A (13.7), and blue dextran 2000
(Vo). Inset indicates the profile of immunoblotting with antiserum
against the 42- and 19-kDa subunits of PR-IP of fractionated material
from a Sephacryl S-300 HR column after SDS-PAGE on a 15% polyacrylamide gel.
Fig. 6 Gel filtration of MS-SP-minus activity on a Sephacryl S-100
HR column. Conditioned medium, in which mt– cells had been cultured was lyophilized and applied to a Sephacryl S-100 HR column
and eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM
NaCl, at a flow rate of 5 ml h–1. Fractions of 2 ml were collected and
assayed for mucilage secretion-stimulating activity. Broken line indicates control secretion level in MI medium. Arrows indicate the elution points of marker proteins (kDa): albumin (66), ovalbumin (45),
chymotrypsinogen A (25), ribonuclease A (13.7), and blue dextran
2000 (Vo). Inset indicates the profile of immunoblotting with antiserum against the PR-IP inducer of fractionated material from a
Sephacryl S-100 HR column after SDS-PAGE on a 15% polyacrylamide gel.
esses, otherwise successful conjugation would not occur.
Detailed analysis concerning on these activities during the sexual reproduction processes is now in progress using the purified
or recombinant pheromones.
Materials and Methods
Table 3 Effects of purified sex pheromones on mucilage
secretion from cells
Cells used for
assay
mt+ cells
mt+ cells
mt– cells
mt– cells
Applied
pheromones
Secreted mucilage
(mg uronic acid / 8´104 cells)
PR-IP inducer
none
PR-IP
none
8.38±0.69
2.65±0.69
12.96±0.83
3.22±0.18
Values are means ± SE.
Strains and vegetative culture
The strains of the heterothallic C. peracerosum-strigosum-littorale complex used in this work were NIES-67 (mt+) and NIES-68 (mt–),
obtained from the National Institute for Environmental Studies (Ibaraki,
Japan). Clonal cultures were grown in 300-ml Erlenmeyer flasks
containing 150 ml of nitrogen-supplemented medium (C medium;
Ichimura 1971) at 24°C under a photoperiodic regime of 16 h of light
and 8 h of darkness. Fluorescent lamps (FL40SS D; Toshiba, Tokyo,
Japan) provided an irradiance of 52 mmol (photons) m–2 s–1 at the surface of the culture medium. Cells were counted in a hemocytometer
and the counts were repeated at least three times to ensure accuracy.
Mating cultures
Vegetative cells from cultures in the late logarithmic growth
phase (14 d after transfer) were harvested by centrifugation and
washed three times with MI medium. Equal numbers of both mating
type cells (6´105 cells each) were mixed in 300-ml Erlenmeyer flasks
1086
Mucilage secretion by Closterium pheromones
that contained 75 ml of MI medium and incubated under continuous
light.
Preparation of secreted mucilage
Cell cultures were thoroughly mixed and filtered through a polycarbonate filter (8 mm; ADVANTEC, Chiba, Japan) to separate the
secreted mucilage from the cells. The filtered media containing mucilage were centrifuged at 10,000´g for 30 min. The precipitates
obtained were washed with distilled water (10,000´g for 30 min),
washed again (45,000´g for 30 min), and finally lyophilized.
Staining of acidic polysaccharides
Alcian blue dye (Wako Pure Chemical Industries, Osaka, Japan)
was dissolved in 3% (v/v) acetic acid to a final concentration of 3%
(w/v), adjusted to pH 2.5, and centrifuged to remove insoluble materials. Cells were cultured for various time periods under continuous
light, at a density of 3´104 cells ml–1 in a 24-well plate, containing
wells of 15.5 mm diameter and 17.3 mm depth (Microplate; Asahi
Techno Grass, Tokyo, Japan). Subsequently, 400 ml of staining solution
were added. After 30 min of staining, the cultures were washed three
times with 400 ml of 3% (v/v) acetic acid, and then suspended to a
final volume of 400 ml in distilled water.
Determination of total sugar content
The amounts of total sugar in the secreted mucilage were determined by the phenol-sulfuric acid method (Dubois et al. 1956) with
glucose as a standard and the carbazole-sulfuric acid method (Bitter
and Muir 1962) with glucuronic acid as a standard, respectively.
Glycosyl composition analysis
Neutral sugars in the mucilage were converted to alditol acetate
derivatives as described by Albersheim et al. (1967) and analyzed by
gas-liquid chromatography with a SP2330 column (0.25 mm ´ 30 m;
Supelco, Belleford, PA, U.S.A.) at 220°C. Neutral and acidic sugars in
the mucilage were determined after conversion to trimethylsilyl ether
of corresponding methylglycosides as described by York et al. (1986).
Samples were analyzed with a DB-1 column (0.25 mm ´ 30 m; J and
W Scientific Co., Folsom, CA, U.S.A.) by gas-liquid chromatography.
Preparation of conditioned medium
mt+ and mt– cells were washed three times with fresh MI medium
and then separately cultured in MI medium at a density of 1.2´l06 cells
per 75 ml in 300-ml Erlenmeyer flasks for 24 h in the light, unless otherwise stated. The cultures were filtrated through qualitative filter
papers (ADVANTEC) to remove cells and mucilage. Then, the filtered
media were used as conditioned media.
Assay of mucilage secretion
mt+ and mt– cells were washed three times with fresh MI medium
and then cultured in MI medium (control) or conditioned medium at a
density of 1.2´l06 cells per 75 ml in 300-ml Erlenmeyer flasks. In
another experiment, 8´l04 cells were cultured in 5 ml of MI medium
that contained aliquots (0.4 ml) of each fraction of gel filtration columns, in 10-ml Erlenmeyer flasks. After 48 h of incubation unless otherwise stated, the cultures were thoroughly mixed, filtered through a
polycarbonate filter and rinsed with distilled water. The filtrates were
centrifuged at 10,000´g for 30 min and the resulting precipitates were
lyophilized. The amount of uronic acid in the resulting mucilage was
determined using the carbazole-sulfuric acid method with glucuronic
acid as a standard. Statistical analysis was done according to standard
procedures wherever necessary. Results represented here are averages
of three independent experiments. However, standard errors are drawn
as a bar on the points.
Gel filtration of MS-SP-plus and MS-SP-minus
Conditioned medium (2,400 ml), in which mt+ or mt– cells
(7.7´107 cells each) had been separately cultured for 48 h was lyophilized, resuspended with 0.8 ml of 50 mM Tris-HCl buffer (pH 8.0)
containing 100 mM NaCl and applied to a Sephacryl S-300 HR
(Amersham Biosciences, Piscataway, NJ, U.S.A.) column (15 mm
diameter, 500 mm long) or the S-100 HR (Amersham) column (15 mm
diameter, 500 mm long), which had been equilibrated with the same
buffer, respectively. Respective columns were eluted with the same
buffer at a flow rate of 5 ml h–1 and fractions of 2 ml were collected
and assayed for MS-SP-plus and MS-SP-minus activities. For the estimation of Mr on the Sephacryl S-300 HR column, thyroglobulin (669),
catalase (232), ovalbumin (45), ribonuclease A (13.7) were used as
marker proteins (kDa). For the estimation of Mr on the Sephacryl S100 HR column, albumin (66), ovalbumin (45), chymotrypsinogen A
(25), ribonuclease A (13.7) were used as marker proteins (kDa). Blue
dextran 2000 was also used as a marker of void volume of these columns.
Assay of PR-IP and PR-IP inducer on the secretion of mucilages from
cells
PR-IP and PR-IP inducer were purified from media in which mt+
(2´106) and mt– (4´105) cells and in which mt+ (4´105) and mt–
(2´106) cells had been co-cultured in 75 ml MI medium for 48 h in
300-ml Erlenmeyer flasks, respectively (Sekimoto et al. 1990, Nojiri et
al. 1995). mt+ and mt– cells were cultured in 10-ml Erlenmeyer flasks
at a density of 8´l04 cells per 5 ml MI medium, in which purified PRIP inducer (5.3´10–10 M) and PR-IP (5.3´10–9 M) were contained,
respectively. After 48 h of incubation, the amounts of uronic acid in
the resulting mucilage were determined.
Electrophoresis
SDS-PAGE was performed using the method of Laemmli (1967).
Each sample was mixed with 100 mM Tris-HCl buffer (pH 6.8) containing 4% (w/v) SDS, 0.04% (w/v) bromophenol blue, 20% (v/v)
glycerol and 12% (v/v) 2-mercaptoethanol, and the mixture was boiled
for 5 min in a water bath. The sample was then centrifuged at
13,000´g for 5 min and the resulting supernatant was subjected to
electrophoresis on a 15% polyacrylamide separation gel.
Immunological detection
Following SDS-PAGE, the proteins in the gel were electrotransferred onto a PVDF membrane filter (BIO-RAD, Hercules, CA,
U.S.A.) in a buffer of 25 mM Tris, 192 mM glycine and 20% (v/v)
methanol at 60 V for 4 h (Gershoni and Palade 1982). The membrane
was incubated with 10% (v/v) newborn calf serum (Sigma, St. Louis,
MO, U.S.A.) in PBS containing 0.2% (v/v) tween 20 (t-PBS) at room
temperature for 1 h. PR-IP was detected on an immunoblot using a
mixture of the anti-42-kDa (Sekimoto et al. 1993) and anti-19-kDa
subunit (Sekimoto 2002) of PR-IP antisera as the primary probe. For
detection of the PR-IP inducer on an immunoblot, anti-PR-IP inducer
antiserum was used as the primary probe (Sekimoto 2002). The secondary probe was a goat anti-rabbit IgG alkaline phosphatase-conjugated antibody. The membrane was immersed for 5 min in ImmuneStar AP substrate (BIO-RAD). Chemiluminescence on the blot was
captured by a Fluor-S MAX imager (BIO-RAD).
Acknowledgment
We thank Dr. T. Ishii and Dr. K. Kakegawa of Forestry and Forest
Products Research Institute for their invaluable suggestions. This work
was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (no. 13024225; Targeted Pursuit of Challenging Bioactive
Mucilage secretion by Closterium pheromones
Molecules) from the Ministry of Education, Science, Sports, and Culture, of Japan, and for the Encouragement of Young Scientists (no.
13740451) from the Japan Society for the Promotion of Science, of
Japan, to H.S.
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(Received May 18, 2003; Accepted August 7, 2003)