Plant Cell Physiol. 46(9): 1472–1476 (2005)
doi:10.1093/pcp/pci158, available online at www.pcp.oupjournals.org
JSPP © 2005
A Sex Pheromone, Protoplast Release-inducing Protein (PR-IP) Inducer,
Induces Sexual Cell Division and Production of PR-IP in Closterium
Yuki Tsuchikane 1, *, Tadashi Fujii 2, Motomi Ito 1 and Hiroyuki Sekimoto 3, 4
1
Department of General Systems Studies, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902
Japan
2
Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193 Japan
3
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902 Japan
4
Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681
Japan
;
The sex pheromone protoplast release-inducing protein (PR-IP) inducer and a sexual cell division-inducing
pheromone-minus (SCD-IP-minus) that mediates the sexual reproduction of the heterothallic Closterium peracerosum-strigosum-littorale (C. psl) complex were investigated in
this study. Recombinant PR-IP inducer produced by yeast
cells was prepared and assayed for production of PR-IP
and induction of SCD. Both biological activities were
observed after treating mating-type plus (mt+) cells with the
recombinant pheromone. SCD was induced by exposure to
a lower concentration of the same pheromone and by a
shorter treatment period with the pheromone than was production of PR-IP. This indicates that the previously characterized PR-IP inducer has both PR-IP-inducing and SCDinducing activities with mt+ cells, although the inducing
mechanisms of the two pheromones differ.
gametes are well characterized. These are volatile, low molecular weight hydrocarbons (Maier 1995). In the green alga Volvox
carteri, a glycoproteinaceous pheromone, which is probably
the most potent biological effector molecule known, acts as a
sexual inducer (Hallmann 2003). In addition, chemotactic pheromones (lurlenic acid and lurlenol) have been reported in the
heterogamous green alga Chlamydomonas allensworthii. These
are derivatives of pentosylated hydroquinones, which are produced by the female gametes to attract the male gametes for
mating (Starr et al. 1995, Jaenicke and Starr 1996).
Keywords: Closterium — Conjugation — Pheromone — Sexual cell division — Sexual reproduction
Abbreviations: C. psl, Closterium peracerosum-strigosum-littorale; MI medium, mating-inducing medium; mt+, mating-type plus;
mt–, mating-type minus; PR-IP, protoplast release-inducing protein;
SCD, sexual cell division; SCD-IP, sexual cell division-inducing pheromone.
Introduction
Sexual reproduction is an essential process for almost all
panmictic populations of organisms. Although each organism
has its own manner of sexual reproduction, there is a common
basic process in which two specialized, sexually competent
cells recognize each other and fertilization or conjugation takes
place between them. For these events to occur, one cell must
have the ability to communicate with the other. Some specific
substances, such as sex pheromones, are involved in the success of this communication.
In brown algae, chemotactic pheromones that are responsible for the attraction of the male gametes toward the female
*
Fig. 1 The effects of various doses of purified recombinant PR-IP
inducer on the production of PR-IP and induction of SCD of mt+ cells.
(A) Immunological detection of the 19 kDa subunit of PR-IP induced
by recombinant PR-IP inducer. (B) The amount of 19 kDa subunit of
PR-IP detected from each dose (filled circles), and the number of
gametangial cells (open circles). The amounts were determined densitometrically from the bands on the blot. Both activities are expressed
relative to the amount obtained with 3×10–7 M recombinant pheromone. The vertical bars indicate the SE (n = 3).
Corresponding author: E-mail, [email protected]; Fax, +81-3-5454-6638.
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Function of sex pheromone in Closterium
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Fig. 2 The effects of incubation duration with purified recombinant PR-IP
inducer on biological activity. (A) Immunological detection of the 19 kDa subunit
of PR-IP induced by recombinant PR-IP
inducer. (B) The 19 kDa subunit of PR-IP
was quantified and expressed relative to
the amount obtained from a 48 h incubation with the pheromone. (C) The number
of gametangial cells was counted 24 h
after beginning the first incubation with
the recombinant pheromone. The vertical
bars indicate the SE (n = 3).
The morphological features of vegetative cells and the
modes of the sexual reproductive processes of Closterium (unicellular charophycean algae) have been well documented
(Cook 1963, Lippert 1967). Recently, sequence tags expressed
during the sexual reproduction of the Closterium peracerosumstrigosum-littorale (C. psl) complex have also been obtained
(Sekimoto et al. 2003).
It has been suggested that several sex pheromones mediate
the sexual reproduction processes (Sekimoto 2000) of the C.
psl complex. Two sex pheromones have been successfully
isolated from the C. psl complex: protoplast release-inducing
protein (PR-IP) and PR-IP inducer. PR-IP inducer is a glycoprotein with a mol. wt of 18,700 (Nojiri et al. 1995). It is
released from mating-type minus (mt–) cells and induces the
production of PR-IP from mating-type plus (mt+) cells
(Sekimoto et al. 1993, Sekimoto et al. 1994, Sekimoto et al.
1998). PR-IP is a glycoprotein consisting of 19 and 42 kDa
subunits, and it induces the release of gametic protoplasts of
mt– cells (Sekimoto et al. 1990).
In Closterium ehrenbergii, the activating substances
involved in sexual cell division (SCD) have been isolated
(Fukumoto et al. 2002). The pheromone-inducing SCD of mt+
cells (an 18 kDa glycoprotein) was named SCD-inducing pheromone (SCD-IP). Its cDNA was cloned and revealed signifi-
cant sequence similarity to the PR-IP inducer of the C. psl
complex (Fukumoto et al. 2003). Recently, biologically active
SCD-IPs specific for each mating type from the C. psl complex have also been detected (Tsuchikane et al. 2003). Partially
purified SCD-IP-minus acting on mt+ cells and SCD-IP-plus
acting on mt– cells have characteristics similar to the PR-IP
inducer and PR-IP, respectively, in terms of their molecular
weight, heat stability and the light dependency of their secretion. This indicates that SCD-IP-minus and the PR-IP inducer
are related morphologically and physiologically.
Using a recombinant PR-IP inducer, we determined the
dose dependency and incubation time required for inducing
SCD- and PR-IP-inducing activities.
Results
When vegetative cells of the mt+ strain (mt+ cells) of the
C. psl complex were incubated in nitrogen-depleted matinginducing medium (MI medium; Ichimura 1971) containing various concentrations of the purified recombinant PR-IP inducer,
greater amounts of PR-IP were produced (Fig. 1A). As shown
in Fig. 1B, the number of gametangial cells increased with the
dose of recombinant PR-IP inducer added to the MI medium.
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Function of sex pheromone in Closterium
Fig. 3 The effects of short durations of various doses of purified
recombinant PR-IP inducer on the production of PR-IP and the induction of SCD of mt+ cells. The 19 kDa subunit of PR-IP was quantified
(filled circles) and expressed relative to the amount obtained with
3×10–7 M recombinant pheromones for a 48 h incubation. The number
of gametangial cells was counted 24 h after beginning the first incubation with the recombinant pheromone (open circles). The SCD-inducing activity was expressed relative to the amount obtained for a 2 h
incubation with 3×10–7 M recombinant pheromone. The vertical bars
indicate the SE (n = 3).
To distinguish the two different activities of the pheromone, mt+ cells were incubated in MI medium with the
recombinant PR-IP inducer for different times, and then transferred to pheromone-free medium. The number of gametangial
cells and the amount of released PR-IP were quantified 24 and
48 h after beginning the treatment with recombinant pheromone. The production of PR-IP increased with the duration of
incubation in MI medium with the recombinant PR-IP inducer
(Fig. 2A, B). In contrast, the maximal number of gametangial
cells was reached after a 2 h incubation with the pheromone
(Fig. 2C). With longer pheromone treatment, the numbers of
gametangial cells decreased gradually.
When the mt+ cells were incubated in MI medium with
various concentrations of purified recombinant PR-IP inducer
for 2 h, SCD was initially induced by 1×10–12 M recombinant
PR-IP inducer, whereas production of PR-IP was not seen with
3×10–7 M of the inducer (Fig. 3).
Discussion
In a previous study, we detected SCD-IPs (SCD-IP-minus
and -plus), which were released from mt– and mt+ cells of the
C. psl complex, and postulated that these were closely related
to PR-IP inducer and PR-IP, respectively (Tsuchikane et al.
2003).
As shown in Fig. 1, the purified recombinant PR-IP inducer had PR-IP-inducing activity. The dose–response curve
Fig. 4 A schematic illustration of the sexual reproduction processes
in the C. psl complex. (A) Vegetative cells of the mt+ and mt– strains;
(B) mucilage (gray circles) secretions; (C) sexual cell division (SCD);
(D) formation of a sexual pair induced by an unknown pheromone; (E)
release of the gametic protoplasts; (F) zygospore formation by fusion
of the cells. Black arrows indicate the interaction of PR-IP and PR-IP
inducer.
was similar to that reported for the native purified PR-IP inducer (Nojiri et al. 1995). Moreover, the recombinant PR-IP
inducer also showed SCD-inducing activity, although the concentration required for induction was slightly lower than that
for PR-IP induction. This indicates that the recombinant PR-IP
inducer stimulates both production of PR-IP and induction of
SCD in mt+ cells, and suggests that SCD-IP-minus and PR-IP
inducer are identical.
As shown in Fig. 2, the amount of PR-IP produced was
dependent on the duration of treatment with the PR-IP inducer.
In contrast, SCD was induced with shorter treatments with the
recombinant PR-IP inducer than required for the production of
PR-IP. SCD was also induced by exposure to very low concentrations of the recombinant PR-IP inducer for short durations
(Fig. 3). These results suggest that the modes of action for the
two activities differ. The simplest explanation of the differences in the actions is the presence of different receptors. Using
a FLAG-tagged recombinant pheromone would be a suitable
way to elucidate these actions.
In the C. psl complex, sexual reproduction consists of the
following processes: (i) a vegetative cell of each mating type
produces two gametangial cells by SCD; (ii) a sexual pair is
Function of sex pheromone in Closterium
formed between the different mating types of gametangial
cells; (iii) each cell of the pair releases a naked cell (protoplast) of the gamete via the interaction of PR-IP and PR-IP
inducer; and (iv) these two cells fuse to form a zygospore (Fig.
4). In addition, secretion of mucilage from each mating-type
cell is induced soon after mixing the two mating types
(Akatsuka et al. 2003). During these processes, SCD in mt+
cells is produced when the concentration of PR-IP inducer is
still very low. The induction of PR-IP production in mt+ cells
requires both the accumulation of PR-IP inducer released from
mt– cells and the action of the pheromone for a period of time.
As shown in Fig. 4, PR-IP and PR-IP inducer were also
responsible for mucilage secretion in mt– and mt+ cells, respectively, although the dose dependency of this has not been
examined (Akatsuka et al. 2003). Furthermore, the induction of
SCD in mt– cells by the action of purified PR-IP has been
tentatively confirmed (S. Akatsuka et al. personal communication). The success of sexual reproduction in Closterium is largely
dependent on exact intercellular communication mediated by sex
pheromones, and this communication is very complex.
Materials and Methods
Strains and culture conditions
The strains NIES-67 (mt+) and NIES-68 (mt–) of heterothallic C.
psl complex used in this study were obtained from the National Institute for Environmental Studies, Environmental Agency (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 16 h light/8 h dark regime. Light from
fluorescent lamps (FL40SSD; Toshiba, Tokyo, Japan) was adjusted to
52 µmol m–2 s–1 at the surface of the culture medium.
Vegetative mt+ and mt– cells of the C. psl complex in the late
logarithmic phase were centrifuged and washed three times with MI
medium. The cells were incubated in MI medium for 24 h under
continuous light and washed again with MI medium before use. These
pre-cultured mt+ cells were used to examine the production of PR-IPand SCD-inducing activity. To induce sexual reproduction, 10,000 precultured cells of the mt+ and mt– strains were each mixed in 2 ml of
MI medium and incubated for 72 h under continuous light (130 µmol
m–2 s–1).
Expression and purification of recombinant proteins
Following the methods of Sekimoto (2002), transformed yeast
cells that secreted FLAG-tagged recombinant PR-IP inducer were prepared. The recombinant yeast cells were cultured in liquid media with
minimal SD base (Clontech, Palo Alto, CA, USA) and with –Trp
Dropout supplement (Clontech) (SDM minus Trp) at 30°C for 24 h.
After a 48 h preliminary culture with shaking, the starter cultures were
diluted 20-fold with YP4 high-stability expression media [YPHSM;
1% (w/v) glucose, 3% (w/v) glycerol, 1% (w/v) yeast extract, 8% (w/
v) peptone, 20 mM CaCl2] and incubated at 30°C for 3 more days,
with continued shaking of the flasks. The cultured cells were harvested by centrifugation for 30 min at 10,000×g and, to remove all
cells, the supernatant was filtered through a membrane filter (Durapore, GVWP, Millipore, Bedford, MA, USA). The filtrate was run
through an ANTI-FLAG M2 affinity gel column (Sigma, St Louis,
MO, USA), equilibrated with Tris-buffered saline. The recombinant
PR-IP inducer was eluted with the same buffer containing 100 µg ml–1
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FLAG peptide (Sigma) at a flow rate of 20 ml h–1. The eluate was subjected to ultrafiltration (Ultrafree-PFL, 5000 NMWL, Millipore) for
desalting and removing the FLAG peptide, and the buffer was
exchanged for MI medium. The protein content was determined using
bovine serum albumin as the standard, according to the method of
Bradford (1976).
Biological activity of recombinant PR-IP inducer in the production of
PR-IP and the induction of SCD
To detect the production of PR-IP from mt+ cells, 50,000 cells,
pre-cultured in MI medium, were incubated in 2 ml of MI medium
containing various concentrations of the FLAG-tagged recombinant
PR-IP inducer for 48 h under continuous light. The medium including
released PR-IP was filtered through a membrane to remove the cells,
and lyophilized. PR-IP was detected by immunoblotting after subjecting the samples to SDS–PAGE.
To detect SCD-inducing activity, 20,000 pre-cultured mt+ cells
were incubated in 2 ml of MI medium with various concentrations of
FLAG-tagged recombinant PR-IP inducer under continuous light.
After incubation for 24 h, the number of gametangial cells was
counted under microscopy.
To examine the effect of the duration of incubation with the
recombinant pheromone, mt+ cells were incubated in MI medium containing 1×10–7 M or various concentrations of purified recombinant PRIP inducer (first culture). After 0, 2, 4, 8, 24 and 48 h, the supernatant
of each culture medium was collected and the resultant cells were
incubated again in 2 ml of fresh MI medium (second culture). The
number of gametangial cells was counted 24 h after beginning incubation with the recombinant pheromone. To detect PR-IP-inducing activity, a second culture medium was collected 48 h after beginning
incubation with the recombinant pheromone, mixed with the respective first culture medium containing recombinant pheromone,
lyophilized, and subjected to SDS–PAGE followed by immunoblotting.
Electrophoresis and immunological detection
SDS–PAGE was performed using the method of Laemmli (1967)
on 15% polyacrylamide separation gels. Each sample was mixed with
an equal volume of 120 mM Tris–HCl buffer (pH 6.8) that contained
4% (w/v) SDS (Wako Pure Chemical Industries, Osaka, Japan), 0.04%
(w/v) bromophenol blue (Wako), 20% glycerol (v/v) and 10% (v/v) 2mercaptoethanol, boiled for 5 min in a water bath, and then subjected
to electrophoresis.
The proteins on the gel were transferred electrophoretically onto
a polyvinylidene difluoride (PVDF) membrane filter (Millipore) in
25 mM Tris, 192 mM glycine and 20% (v/v) methanol in water at 60 V
for 4 h (Gershoni and Palade 1982). The membrane was incubated
with 10% (v/v) newborn calf serum (Dainippon Pharmaceutical,
Osaka, Japan) in phosphate-buffered saline at room temperature for
1 h.
PR-IP was detected on an immunoblot using an anti-19 kDa
subunit of PR-IP antiserum as the primary probe (Sekimoto 2002). The
secondary probe was a goat anti-rabbit IgG alkaline phosphataseconjugated antibody. The membrane was immersed for 5 min in
ImmuneStar AP substrate (BIO-RAD, Hercules, CA, USA). Chemiluminescence on the blot was quantified using Light Capture (ATTO,
Tokyo, Japan).
Acknowledgments
This work was supported by Grants-in-Aid (No. 1610982,
17370087 and 17405013) from the Japanese Society for the Promotion of Science to Y.T. and H.S.
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Function of sex pheromone in Closterium
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(Received January 5, 2005; Accepted June 15, 2005)