Plant Cell Physiol. 46(2): 312–316 (2005)
doi:10.1093/pcp/pci025, available online at www.pcp.oupjournals.org
JSPP © 2005
The Regulatory Networks of Gene Expression during the Sexual
Differentiation of Chlamydomonas reinhardtii, as Analyzed by Mutants for
Gametogenesis
Jun Abe 1, Takeaki Kubo 1, Tatsuaki Saito 2 and Yoshihiro Matsuda 3, 4
1
Department of Molecular Science, Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe, 657-8501 Japan
Department of Applied Science, Faculty of Science, Okayama University of Science, Okayama, 700-0005 Japan
3
Department of Biology, Faculty of Science, Kobe University, Nada-ku, Kobe, 657-8501 Japan
2
;
and Granick 1954), especially ammonium ions (Matsuda et al.
1992), vegetative cells of both mating types differentiate independently into sexually competent gametes and undergo mating
when they are mixed. Light-dependent (LD) strains in C. reinhardtii need light to differentiate into gametes under N-starved
conditions, but light-independent (LI) strains can become competent gametes in the dark (Beck and Haring 1996, Saito et al.
1998).
When N is depleted, the vegetative cells undergo two critical programs (Goodenough 1991, Beck and Haring 1996).
First, they express an N-adaptation program to acclimate to N
starvation through a variety of metabolic changes, including
the synthesis of N-scavenging enzymes (Vallon et al. 1993,
Quesada and Fernández 1994). Secondly, cells express a gamete program that produces cells competent for mating. The
gamete program includes the formation of sex-specific agglutinin proteins responsible for flagellar agglutination at the beginning of mating (Adair et al. 1982, Saito et al. 1985) and the
formation of sex-specific mating structures (which in mt+ gametes contain the gamete membrane protein FUS1) responsible
for fusion of mating partners (Friedmann et al. 1968, Goodenough et al. 1982, Ferris et al. 1996).
Genes induced by N starvation have been analyzed in this
alga (Beck and Haring 1996). For example, NIA1, encoding a
nitrate reductase (Quesada and Fernández 1994), and NCG2,
encoding a urate oxidase type-II enzyme (Merchán et al. 2001),
are involved in the N-adaptation program, while FUS1 encoding a sex recognition glycoprotein (Ferris et al. 1996) and MID
specifying the minus dominance (Ferris and Goodenough
1997) are related to the gamete program. Recently, 18 novel Nstarved gametogenesis (NSG) genes have been cloned by
expressed sequence tag (EST)-based macroarray analysis and
they are placed into three temporal classes of gene expression—early, middle and late—during synchronized gametogenesis (Abe et al. 2004). Many of the NSG genes had annotations,
and the predicted proteins were classified into those involved
in the N-adaptation program or in the gamete program.
To define what types of regulatory networks of transcription exist among the genes for the adaptation program and the
gamete program, analyses of mutations for gametic differentiation are highly desirable. We isolated a conditional mutant,
Cells of Chlamydomonas reinhardtii differentiate into
gametes under conditions of nitrogen (N) starvation,
expressing the genes for the N-adaptation program and the
gamete program. To investigate the regulatory networks of
transcription among the N-starvation-inducible genes, we
examined the gene expression in dif mutants, affecting
gametic differentiation. In a conditional mutant, dif2, the
cells remained ‘vegetative’ at the restrictive temperature,
and the induction of 20 out of 21 genes related to the two
programs was impaired. They were expressed soon after
transfer of the cells to the permissive temperature, in parallel with the acquisition of mating ability. In an unconditional mutant, dif3, the cells could not differentiate into
gametes at all, but the induction of only four genes (FUS1,
NSG3, NSG6 and NSG7) related to the gamete program was
impaired. The results suggest that Dif3 regulates putative
N-starvation signal transduction pathways downstream of a
master regulator, Dif2. We also examined a light-dependent laboratory strain that was unable to become gametes in
the dark. The ‘pre-gametes’ placed in the dark, however,
could induce normally all of the 21 genes, suggesting that
light is required for the gametic differentiation at the translational and/or post-translational levels.
Keywords: Chlamydomonas reinhardtii — Nitrogen starvation-inducible genes — Sexual differentiation mutants — Transcription regulation.
Abbreviations: EST, expressed sequence tag; LD, light dependent; LI, light independent; N, nitrogen; TAP, Tris/acetate/phosphate.
Introduction
Simple, amenable experimental systems such Saccharomyces cerevisiae and Chlamydomonas reinhardtii are excellent
models for studies of the mechanism of sexual reproduction.
The unicellular green alga C. reinhardtii is a haploid organism
and cells are of one of two genetically fixed mating types, plus
(mt+) and minus (mt–). When deprived of nitrogen (N) (Sager
4
Corresponding author: E-mail, [email protected]; Fax, +81-78-803-5709.
312
Transcriptional network in N-starved gametogenesis
313
Fig. 1 Kinetics of gametic differentiation (A) and RNA
accumulation (B and C) in dif2 mutant cells. Synchronously grown wild-type (CC-125 mt+) and dif2 mt+ mutant
cells at the end of the dark period (D-12/L-0 cells) were
transferred to N-free medium and incubated at 35°C for
6 h in the light. In dif2, the temperature was shifted down
to 25°C after 6 h incubation. (A) At the times indicated,
the cells were mixed with tester mt– (8511B) gametes in
equal cell numbers and the mating efficiency determined
after a 30 min incubation. (B) Cell samples for RNA isolation were removed at the times indicated and total RNA
(2.5 µg per dot) was subjected to dot-blot hybridization
analysis using marker genes, NIA1, NCG2 and FUS1, as
probes. The L27a gene (encoding ribosomal protein L27a)
was used as a loading control. (C) Dot-blot analysis was
performed using 18 NSG genes as probes and these data
are graphically displayed with color to represent the relative changes in the expression of each gene. Increases in
mRNA levels are shown as shades of red.
dif2, in the previous study (Saito and Matsuda 1991) and an
unconditional mutant, dif3, in the present study, affecting
gametic differentiation. By using a synchronized system for
gametogenesis (Matsuda et al. 1990, Abe et al. 2004), we analyzed here the expression of three marker genes (NIA1, NCG2
and FUS1) and 18 NSG genes in the two dif mutant cells, comparing it with that in the wild-type cells. We also analyzed gene
expression in LD cells when they were placed in the dark under
conditions of N starvation, comparing it with that in LI cells.
Possible regulatory networks of gene expression during
gametic differentiation under conditions of N starvation and
light/dark are discussed.
Results and Discussion
The dif2 (ts) mutation blocks the expression of almost all genes
related to both the N-adaptation program and the gamete program at restrictive temperature
The dif2 mutant cells remain ‘vegetative’ at restrictive
temperature (35°C) in N-free medium (Saito and Matsuda
1991). Fig. 1A shows the kinetics of gametic differentiation at
35°C in wild-type (CC-125, mt+) and dif2 (mt+) mutant cells
under light conditions. When synchronized vegetative cells
were transferred to N-free medium and incubated at 35°C,
wild-type cells developed their agglutinability and fusing ability more rapidly than those incubated at 25°C (Fig. 2) and
reached fully competent gametes after 2 h incubation. In contrast, dif2 mutant cells were unable to agglutinate or fuse when
mixed with tester mt– gametes even after 6 h incubation at
35°C and developed to gametes after a subsequent temperature
shift to 25°C (permissive temperature) within 3 h (Fig. 1A).
To examine the transcriptional activation of three marker
genes, NIA1, NCG2 and FUS1, during gametogenesis, total
RNA was isolated from the wild-type and dif2 mutant cells collected at a 0, 1, 3 and 6 h incubation at 35°C in N-free medium
and used for dot-blot hybridization analyses, using the cDNA
fragments of these genes as specific probes (Abe et al. 2004).
As for dif2 mutant, the cells were transferred from 35 to 25°C
at 6 h and cell samples were collected after 1 h (i.e. at 7 h in Nfree medium) and 3 h (at 9 h) incubation and examined for
gene expression at the permissive temperature.
As shown in Fig. 1B, wild-type cells placed at 35°C transiently accumulated NIA1 and NCG2 mRNA within 1 h and
FUS1 mRNA within 3 h after transfer to N-free medium, this
time course being similar to that in the cells placed at 25°C
(Fig. 2B). In dif2, however, only the NCG2 mRNA was accumulated at 35°C and the accumulation was sustained through
the rest of the time course even after transfer to 25°C. The
NIA1 and FUS1 mRNAs were accumulated only after transfer
to 25°C and incubation for 1 and 3 h, respectively. The L27a
gene, encoding a Chlamydomonas 60S ribosomal protein,
showed a relatively constant level of expression in both cells
placed at 35°C (Fig. 1B).
We also analyzed the steady-state levels of mRNAs for 18
NSG genes by dot-blot analyses and displayed the measured
changes in a graphical format. The results (Fig. 1C) showed
that the dif2 mutation blocks the expression of all of the 18
NSG genes at the restrictive temperature, and that they are
expressed between 1 and 3 h after transfer to the permissive
temperature. It was noticed that NSG17, an N-starvation-specific, early gene encoding a putative transcription factor with a
basic helix–loop–helix DNA-binding domain (Abe et al. 2004),
is the first to be expressed transiently after transfer to 25°C
(Fig. 1C). This suggests that there might be close relationships
between DIF2 and NSG17 in the gene expression cascade.
314
Transcriptional network in N-starved gametogenesis
the activities of cell body agglutinin (Saito et al. 1985) and
gametolysin (Matsuda and Kubo 2004). Neither activity was
contained in the soluble fraction, suggesting that this mutant
belongs to class I (Saito et al. 1988).
As shown in Fig. 2, the mutant cells could express NIA1,
NCG2 (Fig. 2B) and 15 NSG genes (NSG1, NSG2, NSG4,
NSG5 and NSG8–NSG18; Fig. 2C) normally. No transcripts
were detected for FUS1 (Fig. 2B), NSG3, NSG6 or NSG7 genes
(Fig. 2C). We found (Abe et al. 2004) that these four genes are
expressed specifically during gametogenesis of wild-type cells,
since no transcripts are detected in the vegetative cell cycle,
and that the expression occurs at the middle stage (between 3
and 4 h after transfer to N-free medium) in parallel with the
acquisition of mating competency, suggesting their involvement in the gamete program.
Fig. 2 Kinetics of gametic differentiation (A) and RNA accumulation (B and C) in dif3 mutant cells. Synchronously grown wild-type
(CC-125 mt+) and dif3 mt+ mutant cells at the end of the dark period
were transferred to N-free medium and incubated at 25°C for 6 h in the
light. Dot-blot analysis was performed as in Fig. 1.
The dif3 mutation blocks only the expression of four genes related to the gamete program
Newly isolated mutant cells for gametic differentiation,
dif3 mt+, could not develop the ability to agglutinate (data not
shown) or fuse (Fig. 2A) under conditions of N starvation at
25°C. To confirm that dif3 is really a class I mutant that prevents cells from differentiating into gametes (Saito et al. 1988),
the cells were harvested after 6 h incubation in N-free medium,
homogenized to prepare the soluble fraction and assayed for
The light-dependent cells for gametogenesis placed in the dark
express all genes related to both the N-adaptation program and
the gamete program
Fig. 3A shows the time course of gametic differentiation in
an LD strain, CC-1690 mt+, and an LI strain, CC-125 mt+ (Saito
et al. 1998), when the synchronized vegetative cells at the early
G1 stage were transferred into N-free medium and incubated in
the dark at 25°C. Both cells placed in the light developed mating ability after 1 h incubation, with maximal activity being
reached after 3–4 h (data not shown). In the dark (Fig. 3A), no
gametes were detected in LD cells even after 6 h incubation,
while LI cells developed to gametes with a similar time course
to that of the cells placed in the light (Fig. 2A). When the LD
cells were exposed to light after 6 h incubation in the dark, they
became fully competent gametes within 1 h (Fig. 3A).
To investigate whether the gametogenetic genes are
expressed in the LD cells placed in the dark, the steady-state
Fig. 3 Kinetics of gametic differentiation (A) and
RNA accumulation (B and C) in LD (CC-1690 mt+)
and LI (CC-125 mt+) cells. Synchronously grown
cells at the end of the dark period were transferred
to N-free medium and incubated at 25°C for 6 h in
the dark. The LD cells were exposed to light after
6 h dark incubation. Dot-blot analysis was performed as in Fig. 1.
Transcriptional network in N-starved gametogenesis
Fig. 4 Schematic diagram of the regulatory networks of gene expression during gametic differentiation under conditions of N starvation.
levels of mRNAs were measured, comparing them with those
in the LI cells. The results (Fig. 3B, C) showed that the LD
cells could express all of the 21 genes normally in the dark. We
also found that the transcripts for many of these genes are
decreased during 6 h incubation in the dark and accumulated
again, albeit slightly, when the dark-incubated LD cells were
transferred to the light (Fig. 3B, C). The accumulation was
detected between 1 and 5 h in the light, a time after the completion of gametic differentiation (Fig. 3A), suggesting that the
light-induced transcripts are not directly involved in the formation of sexually competent gametes. It was noticed that the
transcriptional levels of the L27a gene were significantly
increased when the LD cells were transferred from the dark to
the light (Fig. 3B), suggesting that the activation of genes may
occur more generally by exposure to light.
Beck and Acker (1992) reported that N starvation in the
dark induces LD cells to pre-gametes and they are converted
into gametes upon light illumination. Light has been shown to
act at a late stage of gametic differentiation (Treier et al. 1989,
Beck and Haring 1996). Saito et al. (1998) showed that the pregametes fail to accumulate cell body agglutinins or to form
mating structures and that these essential components for gametes are constructed upon exposure to light. Since we found in
the present study that the pre-gametes can transcribe the genes,
even if not all of them, for N-starved gametogenesis, light
might be needed for the program of gametogenesis at their
translational or post-translational levels. A preliminarily experiment showed that the addition of 10 µg of cycloheximide ml–1,
an inhibitor of protein synthesis, to the pre-gametes (CC-1690)
1 h before light exposure (at 5 h in the dark) almost completely
blocked their subsequent steps to agglutinate and fuse in the
light. It will be of interest to examine whether the proteins for
gamete-specific genes such as FUS1, NSG3, NSG6 and NSG7
are produced in the pre-gametes.
315
Regulatory networks of gene expression in cells placed under
N-starved conditions
Fig. 4 shows a possible expression cascade of N-starvation-inducible genes examined in the present study. Since all of
the 21 genes were expressed in the pre-gamete state in the LD
strain that requires light for gamete formation (Fig. 3), light
might not commit them to the gene expression cascade at all.
We anticipate that N starvation inside the Chlamydomonas
cells may induce the expression of the DIF2 gene and that the
product Dif2 can then act as a positive regulator of almost all
genes, which are related both to the N-adaptation program and
the gamete program. With only the exception of NCG2 encoding a urate oxidase II (Merchán et al. 2001), the dif2 mutation
impaired the expression of NIA1, FUS1 and all of the 18 NSG
genes examined (Fig. 1), suggesting that Dif2 is a possible regulator located upstream in the gene expression cascade.
The dif3 mutation impaired the expression of only four
genes, FUS1, NSG3, NSG6 and NSG7 (Fig. 2), suggesting that
Dif3 is probably a regulatory factor located at the position
lower than Dif2 in the N-starved signal transduction cascade
(Fig. 4). Except for FUS1 (Misamore et al. 2003), the precise
functional roles of NSG3, NSG6 and NSG7 for gametic differentiation and mating are still unclear at present: NSG3 has only
been annotated as a putative reverse transcriptase, NSG6 as a
novel soluble protein with no significant homology to any database, and NSG7 as a novel protein with a motif of ankyrin
repeats (Abe et al. 2004). Identification of these NSG proteins
and also Dif2 and Dif3 regulators would enhance our understanding of the molecular mechanism of gametic differentiation under conditions of N starvation.
Materials and Methods
Strains
Laboratory strains of C. reinhardtii, 21 gr mt+ (CC-1690), CC125 mt+, 8511A mt+ and 8511B mt– were used in this study (Saito et al.
1998). The conditional mutant for gametic differentiation, dif2 mt+, is
that isolated previously (Saito and Matsuda 1991). An unconditional
mutant for gametic differentiation, dif3 mt+, was isolated according to
the methods described previously (Saito et al. 1988, Saito and Matsuda 1991). Vegetative cells of sr1 mt+ grown on Tris/acetate/phosphate (TAP) medium (Gorman and Levine 1965) were exposed to UV
light (3,800 erg cm–2 s–1) for 90 s to obtain about 0.1% survival. The
mutagenized cells were grown for 1 d in the dark and 3 d in the light.
The cells were then transferred to N-free medium (see below), incubated at 25°C for 7 h to induce gametes, mixed with an excess of arg2
mt– gametes and allowed to mate overnight. The pellicle of the zygotes
was removed and the remaining single cells were spread on TAP plates
containing 10 µg ml–1 streptomycin sulfate to kill all mt– cells and
most of the remaining zygotes (they are sensitive to streptomycin as
the sr1 is recessive to SR1 in diploids). The colonies that developed
were isolated and re-tested for their mating ability at 25°C. To select
class I mutants that prevent cells from differentiating to gametes under
conditions of N starvation (Saito and Matsuda 1991), the non-mating
clones were cultured in N-free medium for 14 h, homogenized to prepare the soluble fraction and assayed for cell body agglutinin and
gametolysin activity. One clone that showed neither activity appeared
to belong to class I and was designated as dif3.
316
Transcriptional network in N-starved gametogenesis
Gamete induction
Cells were synchronized in minimal (M) salt medium containing
3.7 mM ammonium nitrate as a sole nitrogen source (Sager and Granick 1953) under a cycle of 12 h light and 12 h dark (Matsuda et al.
1995). Synchronized vegetative cells at the early G1 stage (i.e. cells at
the end of the dark period, D-12/L-0 cells) were harvested by centrifugation at 700×g for 2 min, washed twice in M-N (N-free) medium,
resuspended in the medium at about 1.2×107 cells ml–1 and incubated
at either 35 or 25°C in the light (3,000 lux) (Saito and Matsuda 1991).
Gametic differentiation of cells was monitored by mixing them in
equal numbers with tester gametes of the opposite mating type. The
tester gametes of mt+ (8511A) and mt– (8511B) were obtained from
plate cultures: cells were grown for 6 d on 1.5% agar plates containing
TAP, then flooded in N-free medium at about 1×107 cells ml–1 and
incubated for 2–3 h (Saito et al. 1988). The flagellar agglutinability
was scored within 5 min after mixing and the mating efficiency (the
cell fusion) was determined after 30 min incubation in the light at
either 35 or 25°C (Matsuda et al. 1978).
For induction of gametes in the dark, the synchronized D-12/L-0
cells were harvested, washed, resuspended in N-free medium in a dark
room with minimum exposure to a dim red light and incubated in the
dark with gentle shaking (Saito et al. 1998). Mating competence of
cells was determined by mixing in the dark with tester gametes
(induced in the light) of the opposite mating type.
Dot-blot analysis
Total RNAs were extracted from about 1×109 cells taken at different time points of gametic induction in N-free medium by the methods described by Kubo et al. (2001). For dot-blot analysis, aliquots of
2.5 µg of total RNA were applied to Hybond-N+ membranes (Amersham) and hybridized with gene probes (Sambrook and Russell 2001).
The gene probes for NIA1 (Fernández et al. 1989), NCG2 (Pozuelo et
al. 2000), FUS1 (Ferris et al. 1996), L27a and NSG1–NSG18 (Abe et
al. 2004) were prepared as described previously (Abe et al. 2004). The
hybridization profiles of each gene were shown in a tabular form using
Cluster and TreeView (Eisen et al. 1998).
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(Received September 17, 2004; Accepted November 28, 2004)