Mating-Induced Shedding of Cell Walls, Removal of
Walls from Vegetative Cells, and Osmotic Stress Induce
Presumed Cell Wall Genes in Chlamydomonas1
Xenia-Katharina Hoffmann and Christoph F. Beck*
Institute of Biology III, Albert-Ludwigs-University, D–79104 Freiburg, Germany
The first step in sexual differentiation of the unicellular green alga Chlamydomonas reinhardtii is the formation of gametes. Three
genes, GAS28, GAS30, and GAS31, encoding Hyp-rich glycoproteins that presumably are cell wall constituents, are expressed
in the late phase of gametogenesis. These genes, in addition, are activated by zygote formation and cell wall removal and by
the application of osmotic stress. The induction by zygote formation could be traced to cell wall shedding prior to gamete fusion since it was seen in mutants defective in cell fusion. However, it was absent in mutants defective in the initial steps of
mating, i.e. in flagellar agglutination and in accumulation of adenosine 3#,5#-cyclic monophosphate in response to this agglutination. Induction of the three GAS genes was also observed when cultures were exposed to hypoosmotic or hyperosmotic
stress. To address the question whether the induction seen upon cell wall removal from both gametes and vegetative cells was
elicited by osmotic stress, cell wall removal was performed under isosmotic conditions. Also under such conditions an
activation of the genes was observed, suggesting that the signaling pathway(s) is (are) activated by wall removal itself.
Environmental signals control differentiation and
development in plants. In the unicellular green alga
Chlamydomonas reinhardtii, a plant model organism,
starvation for a utilizable nitrogen source initiates the
sexual life cycle that results in the formation of zygotes
(Sager and Granick, 1954; Kates and Jones, 1964; Beck
and Acker, 1992; Matsuda et al., 1992). For completion
of gamete formation, blue light as a second extrinsic
cue is required. This signal becomes effective in the
late phase of sexual differentiation (Treier et al., 1989;
Weissig and Beck, 1991; Beck and Acker, 1992). Irradiation with blue light also is required to maintain the
gametes’ mating competence (Pan et al., 1997).
The initial contact between gametes of mating types
plus and minus is mediated by the mating-typespecific agglutinins, high Mr glycoproteins on the flagellar surface (Adair et al., 1983; Ferris et al., 2005). The
resulting flagellar adhesion generates signaling pathways that activate the interacting cells for cell-cell
fusion. A consequence is the activation of flagellar adenylyl cyclase, resulting in a nearly 10-fold increase of
intracellular cAMP in each of the interacting gametes
(Pasquale and Goodenough, 1987; Saito et al., 1993).
This triggers dramatic alterations in the matingcompetent cells (for review, see Pan and Snell, 2000a),
among them the release of a matrix-degrading pro1
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft to C.F.B. X.-K.H. acknowledges the support by the Deutscher Akademischer Austauschdienst for a stay in
the laboratory of Dr. Purton.
* Corresponding author; e-mail [email protected]; fax 49–
761–203–2745.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.105.065037.
tease named gamete lytic enzyme (GLE) that degrades
the gametes’ cell wall (Matsuda et al., 1987; Snell et al.,
1989; Kinoshita et al., 1992) and the erection of apically localized mating structures (Cavalier-Smith, 1974;
Goodenough and Weiss, 1978).
Fusion of gametes after initial contact of the mating
structures triggers another signaling cascade that,
among other events (reviewed in Pan and Snell,
2000a), manifests itself in the up-regulation of genes.
Some of these genes expressed in young zygotes encode
cell wall proteins (Woessner and Goodenough, 1989;
Matters and Goodenough, 1992; Suzuki et al., 2000).
Zygotes elaborate a thick wall that ensures the cells’
survival under adverse environmental conditions,
such as desiccation and low temperatures (Minami
and Goodenough, 1978; Harris, 1989; Beck and Haring,
1996). This wall is structurally and biochemically
distinct from that of vegetative cells or gametes to
which it bears little resemblance (Grief et al., 1987;
Woessner and Goodenough, 1989). The major constituent of both proteinaceous walls, though, are Hyp-rich
glycoproteins (HRGPs; Catt, 1979; Goodenough and
Heuser, 1985; Goodenough et al., 1986). Different sets
of genes that encode HRGPs are expressed in vegetative cells and zygotes, accounting for the differences
observed (Ferris and Goodenough, 1987; Woessner
and Goodenough, 1989; Uchida et al., 1993; Woessner
et al., 1994). Repetitions of Pro residues are characteristic for HRGPs both in vegetative cells and zygotes
(Woessner and Goodenough, 1989; Suzuki et al., 2000)
as documented by an antibody directed against a
(serpro)10 epitope. This motif was also observed in cell
wall glycoproteins of C. eugametos and Volvox carteri,
other members of the volvocales family (Woessner
et al., 1994).
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Hoffmann and Beck
In activated gametes, shedding of the cell wall is a
dramatic event, resulting in the exposure of naked
protoplasts to the environment. This, under hypoosmotic conditions, will result in water influx due to turgor reduction. A crucial role for maintenance of cell
integrity under these conditions may be assigned to
the contractile vacuole that primarily appears to serve
as a pump to remove excess water entering the cells by
osmosis (Luykx et al., 1997a, 1997b).
In Chlamydomonas, hyperosmotic conditions induce the intracellular accumulation and excretion of
glycerol. This was traced to an activation of enzymes
involved in glycerol production (León and Galván,
1995). Most prominent are results showing that osmotic stress in Chlamydomonas rapidly activates
several phospholipid-based signaling pathways that
result in the accumulation of phospholipids that may
serve as second messengers. Among these compounds
are phosphatidyl-D-inositol 3,5-bisphosphate, lysophosphatidic acid, phosphatidic acid, and diacyl glycerol (Meijer et al., 1999, 2001, 2002; Munnik et al., 2000).
How osmotic stress induces the formation of these
compounds in Chlamydomonas has not yet been elucidated, primarily due to the fact that osmosensors have
not been defined in this organism. In higher plants, the
first osmotic stress receptor identified is the hybridtype His kinase Arabidopsis thaliana histidine kinase 1
(ATHK1). The function of this protein was demonstrated using transformants of a yeast mutant defective in osmosensor kinase synthetic lethal of N-end
rule 1 (SLN1) with an ATHK1 expressing clone that
lead to suppression of the mutant phenotype (Urao
et al., 1999).
In higher plants, similar to the situation in yeast,
hyperosmotic and hypoosmotic stress were shown
to activate mitogen-activated protein (MAP) kinases
(Cazalé et al., 1999; Munnik et al., 1999). In the signaling of the osmotic stress response, a link between
the accumulation of lipid-derived secondary messengers and the activation of MAP kinases is still missing
(Munnik and Meijer, 2001). In algae, an analysis of
osmo-controlled signaling pathways was hampered
by a lack of genes that respond to changes in the osmotic environment.
In this study, we report on three osmo-regulated
genes of C. reinhardtii. These genes, encoding HRGPs,
are activated in the late phase of sexual differentiation.
They experience a second activation that is triggered
by wall shedding upon gamete activation. This latter
activation appears to be mediated by wall removal
since it is also observed when the walls are lost under
isosmotic conditions.
RESULTS
Characterization of Three Genes Expressed Late
during Gametogenesis
We previously reported on genes that are expressed
during sexual differentiation of C. reinhardtii (von
Gromoff and Beck, 1993; Rodriguez et al., 1999). In
those studies, based on cDNA clones, we identified
two genes, GAS28 and GAS29, that are up-regulated in
the late phase of gametogenesis. Here, we characterized the gene structure of GAS28 (GenBank accession
no. DQ017908) and, in addition, report on two new
genes named GAS30 (GenBank accession no. DQ017907)
and GAS31 (GenBank accession no. DQ017909; Fig. 1).
GAS30 exhibits distinct homology to GAS28: with the
exception of parts of its 5# untranslated region (UTR),
the first 160 bp after the translation start codon, and its
3# UTR, GAS30 is nearly identical to GAS28 (Fig. 1B). A
comparison of GAS28 and GAS30 genomic sequences
with sequence information from GAS28 cDNAs as
well as RT-PCR experiments revealed that GAS28
and GAS30 each harbor three exons and two introns,
Figure 1. GAS28, GAS30, and GAS31 gene structures and homology
between predicted gene products. A, Arrangement of GAS28 and
GAS30 within the C. reinhardtii genome. B, Structures of the GAS28,
GAS30, and GAS31 genes. Solid lines indicate 5# and 3# UTRs. Black
boxes indicate deduced protein coding exons; hatched boxes indicate
sequences encoding ser(pro)x-rich domains; dotted lines indicate
introns. Homology between segments of GAS28 and GAS30 sequences
is given in percentage of identity. C, The deduced amino acid
sequences of GAS28, GAS30, and GAS31 are grouped in four domains.
The boxes indicate the different predicted protein domains (numbered
one to four): the secretion signals (dark gray boxes), the N-terminal
globular domain, the ser(pro)x-rich domain (S/P), and the C-terminal
globular domain. The numbers of amino acids in each domain are
given within the boxes. Asterisks indicate potential sites for N-linked
glycosylation. Homology between GAS28 and GAS30 domains is
given in percentage of amino acid identity.
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Wall Shedding and Osmotic Stress Induce Cell Wall Genes
with the exon-intron borders typical for C. reinhardtii
(Silflow, 1998; Fig. 1B). In both GAS28 and GAS30, the
5# UTRs are interrupted by introns. The location of
the exon-intron borders of the second intron in both
genes is identical. Except for an insertion of 772 bp in
the center of the second intron of GAS30, the sequences
of both introns are nearly identical. Genes GAS28 and
GAS30 are arranged as an inverted tandem located
on scaffold 48 of version 2 of the Chlamydomonas
genome sequence (http://genome.jgi-psf.org/chlre2/
chlre2.home.html): the 3# ends of both genes face each
other, separated by about 12 kb (Fig. 1A).
GAS31 was identified in a bacterial artificial chromosome (BAC) clone that hybridized with a probe
derived from the 3# end of a cDNA named GAS29
(Rodriguez et al., 1999). The 3# end of GAS31, starting
at the 3# side of the sequence encoding a ser(pro)x-rich
domain, is identical to GAS29 cDNA. Its 5# end clearly
differs from the GAS29 sequence. GAS31 is located
on scaffold 320 (genome version 2.0). Comparison of
the GAS31 genomic sequence with GAS31 cDNA
sequences revealed that this gene is composed of three
exons and two introns, with the exon-intron borders
typical for C. reinhardtii (Silflow, 1998). Both introns of
GAS31 are located within the coding region in front
of the region encoding the ser(pro)x-rich domain
(Fig. 1B).
We previously identified a cDNA named GAS29
(Rodriguez et al., 1999). The 5# end of the GAS29
cDNA (5# UTR and the first 135 bp after the start of
translation) is identical to that of GAS30, while the 3#
end corresponds to GAS31. A GAS29 gene, however,
could not be identified, neither by screening different
genomic libraries nor by inspection of C. reinhardtii
genomic sequences. Rehybridization of the cDNA library used earlier (Rodriguez et al., 1999) with the 3#
end of GAS29 cDNA revealed an additional GAS29
cDNA clone with the same sequence but a poly(A) tail
that is six bases shorter. To test whether this cDNA
arose by trans-splicing, we attempted to amplify a
GAS29 mRNA using RT-PCR with primers specific for
GAS29. Since no PCR products were obtained, we
cannot decide whether trans-splicing or artificial joining of cDNAs from the 5# end of GAS30 and the 3# end
from GAS31 during the generation of the cDNA library may have generated the GAS29 cDNAs.
Analysis of Predicted Gene Products
The amino acid sequences of the deduced gene
products revealed that each harbors a ser(pro)x-rich
domain, indicative for cell wall proteins (Fig. 1C). The
Pro residues of such domains of extracellular proteins
usually are hydroxylated and, via O-glycosylation, a
substrate for the attachment of sugar residues (O’Neill
and Roberts, 1981; Woessner and Goodenough, 1989;
Vallon and Wollman, 1995). Computer predictions using the BCM program (http://searchlauncher.bcm.
tmc.edu/seq-util/seq-util.html) indicated that the
three proteins are made up of four regions typical for
cell wall glycoproteins of C. reinhardtii (Goodenough
et al., 1986; Woessner and Goodenough, 1992): (1) a
leader sequence that encodes a secretion signal (predicted with TargetP), (2) an N-terminal region, (3)
a ser(pro)x-rich domain, and (4) a C-terminal region
(Fig. 1C). Computer-predicted secondary structures
for domains 2 and 4 using the algorithm HNN (http://
npsa-pbil.ibcp.fr) by Guermeur et al. (1999) revealed
that both domains of all three gene products contain
a-helices, b-strands, and a high percentage (55% to
65%) of random coils. This suggests that domains 2
and 4 of the three proteins may adopt globular configurations. All three proteins also harbor potential
sites for N-linked glycosylation (Fig. 1C). GAS28 and
GAS30 have two sites each, one in domain 2 and one in
domain 4. GAS31 has two sites in domain 2 and three
sites in domain 4. The deduced gene products of
GAS28 and GAS30 are nearly identical in size and
sequence (423 amino acids with a molecular mass of
46.2 kD and 451 amino acids with a molecular mass of
47.8 kD, respectively), whereas the GAS31 protein is
clearly different in size (618 amino acids with a molecular mass of 68.8 kD) and sequence, although it shares
the four protein domains (Fig. 1C).
Presence of Additional Genes with Homology to GAS28,
GAS30, and GAS31 in the C. reinhardtii Genome
We investigated whether additional genes that may
encode proteins related to GAS28 and GAS30 might be
present in the C. reinhardtii genome. For this purpose,
we blasted the deduced amino acid sequences of
GAS28 and GAS30 against version 2.0 of the genomic
sequence using the tBLASTn program. Four different
predicted genes that may encode proteins with similarity to domains 2 and 4 of GAS28 and GAS30 (Fig.
1B) were observed in version 2 of the genome sequence: gene models C_ 70040, C_170162, C_700008,
and C_700009 (representing a single gene) and
C_1550010 (Table I). The gaps in the genomic sequence
from two gene models (C_170162 and C_700008/
C_700009) are located in regions that have a high GC
content (Grossman et al., 2003) and may encode Prorich domains. The alignments of the deduced amino
acid sequences (prediction with BCM) from these gene
models and GAS28 and GAS30 are given in Figure 2
for the N-terminal part (corresponding to domain 2 in
Fig. 1B) and the C-terminal part (corresponding to
domain 4 in Fig. 1B), respectively. Regions that show
a high frequency of conserved residues were numbered one to nine (Fig. 2). These regions are observed
in both, the N-terminal and C-terminal parts of the
predicted proteins and may represent important motifs for the formation of three-dimensional structures.
Except for region four in the C-terminal part and region five in both parts, all conserved motifs have one
or two Cys residues. Because the conserved motifs occur in both parts of the proteins, the three-dimensional
structures of the N-terminal and C-terminal parts are
envisioned to be similar. Additional conserved regions
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Hoffmann and Beck
Table I. GAS28 and GAS30 homologous genes within the C. reinhardtii genome
GAS28 and GAS30 proteins were aligned against predicted products of gene models from the C. reinhardtii draft genome (http://genome.jgi-psf.org/
chlre2/chlre2.home.html; version 2.0) using the tBLASTn program. Gene models with homology to GAS28 and GAS30 are listed, and their locations
within the scaffolds are given. Gene models C_700008 and C_700009 refer to a single predicted gene and therefore are listed together: C_700008
represents its 5# end and C_700009 represents its 3# end. Sequences of the gene models were aligned against sequences from expressed sequence tag
(EST) libraries (www.kazusa.or.jp/en/plant/chlamy/EST and www.biology.duke.edu/chlamy_genome/). The sequences of expressed sequence tag
clones allowed the localization of introns and their borders. Sequence gaps within the regions homologous to GAS28 and GAS30 are listed. n.d., not
determined.
Gene Model
C_70040
C_170162
C_700008/C_700009
C_1550010
Scaffold
7
Location of
Homologous
Regions within
Scaffolds
Gaps within
the Genomic
Sequence
266899–268219
None
17
65789–63307
65095–63955
70
155
191460–192908
64052–65091
191957–192631
64799–64863
were observed that only occur either in the N-terminal
(motifs a and b) or in the C-terminal (motifs c and d)
domains (Fig. 2).
No genes homologous to GAS31 were detected
within the C. reinhardtii genome. We next tested
whether GAS28, GAS30, and GAS31 may exhibit similarity to C. reinhardtii cell wall glycoproteins GP1, GP2
(Goodenough et al., 1986; Ferris et al., 2001; GP2
GenBank accession no. AY596305), VSP3 (Woessner
et al., 1994), class IV cDNA coding for a 34-kD polypeptide (Ferris and Goodenough, 1987; Woessner and
Goodenough, 1989), and ZSP-2 (Suzuki et al., 2000).
No similarities in the amino acid sequences of the
N-terminal or the C-terminal domain were detected,
although these proteins are known to be composed of
globular domains with Pro-rich stretches (Goodenough
et al., 1986; Woessner et al., 1994; Ferris et al., 2001).
Additionally, we tested whether GAS28, GAS30,
and GAS31 may be related to known glycoproteins of
the extracellular matrix from Volvox. The N-terminal
parts (Fig. 1B, domain 2) of these genes revealed a significant similarity to the N-terminal parts of extracellular matrix glycoproteins from Volvox: Pherophorin I,
Pherophorin II, and the sulfated surface glycoprotein
185 (SSG 185; Fig. 3). Pherophorins at their C-terminal
ends show homology to the sexual pheromone from
Volvox (Sumper et al., 1993; Godl et al., 1995), while
glycoprotein SSG 185 forms an insoluble structure
within the extracellular matrix (Ertl et al., 1989). All
three proteins are localized in the cellular zone of the
spheroids (Ertl et al., 1989; Godl et al., 1995). The alignment indicates that the proteins exhibit similarities at
six positions in their N-terminal regions (Fig. 3). These
conserved motifs not only occur in the GAS28, GAS30,
and GAS31 proteins but also in those of the gene models analyzed in Fig. 2. New regions with similarity between these proteins could not be detected. The results
indicate that GAS28, GAS30, GAS31, and the extracellular matrix proteins from Volvox, Pherophorin I,
Predicted
Introns
I: 267215–267400
II: 267711–268029
I: 65782–65614
II: 63518–63336
n.d.
I: 64715–64897
Region
Covered by
ESTs
Yes
Yes
No
Yes, starting from 64271
Pherophorin II, and SSG 185 may share similar threedimensional structures at their N-terminal domains.
Expression Pattern of GAS28, GAS30, and GAS31
during Gametogenesis and in the Early Phase of
Zygote Formation
An accumulation of GAS28 mRNA in the late phase
of gametogenesis has been described previously (von
Gromoff and Beck, 1993; Rodriguez et al., 1999; Abe
et al., 2004). GAS30 and GAS31 showed kinetics of
mRNA accumulation similar although not identical to
those of GAS28 (Fig. 4). The mRNA levels of all three
genes increased in the late phase of gametogenesis.
The expression patterns were compared to those of
FUS1, which encodes a sex recognition protein expressed in the middle phase of gametogenesis (Ferris
et al., 1996; Abe et al., 2004). The strong accumulation
of the FUS1 mRNA in cells 6 h after start of nitrogen
starvation correlated with the appearance of gametes.
Compared to FUS1, the expression of GAS28, GAS30,
and GAS31 was delayed.
The analysis of changes in GAS28, GAS30, and
GAS31 mRNA levels upon zygote formation revealed
an additional mRNA accumulation in young zygotes
(Fig. 4). Only 5 mg/lane of total RNA from young zygotes was applied per lane in contrast to 10 mg/lane of
total RNA from mature gametes. GAS31 mRNA levels
increased rapidly and strongly within 1 h after fusion,
followed by a gradual decrease. The increase in GAS28
and GAS30 mRNA levels was lower and delayed when
compared to the GAS31 transcript. An mRNA accumulation up to 3 h after cell fusion was observed for these
genes. The FUS1 mRNA, in agreement with previous
reports, disappeared in young zygotes (Ferris et al.,
1996), an indication that zygotes were formed. We conclude that the mRNAs of all three GAS genes accumulate not only during the late phase of gametogenesis
but also strongly in young zygotes.
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Wall Shedding and Osmotic Stress Induce Cell Wall Genes
Figure 2. Predicted gene models within the C. reinhardtii genome that may encode proteins with homology to GAS28 and
GAS30. N-terminal and C-terminal domains from GAS28 and GAS30 were BLAST searched against sequences of the current
draft release, version 2.0, of the C. reinhardtii genome (http://genome.jgi-psf.org/chlre2/chlre2.home.html) using the program
tBLASTn. Gene models listed in Table I were used for the alignment. A, Alignment of domain 2 (Fig. 1B) of GAS28 and GAS30
against products of predicted genes of the C. reinhardtii genome. B, Alignment of domain 4 (Fig. 1B) of GAS28 and GAS30
against products of predicted genes of the C. reinhardtii genome. Sequences of gene models obtained from the BLAST search
were translated with BCM six frame translation utilities, aligned using ClustalW, and refined manually. Sequences used for
translation are described in Table I and the text. Residues highlighted in black are conserved in all proteins; those highlighted in
gray are conserved in three or more of the proteins. Conserved amino acids are N/Q, D/E, R/K, S/T, F/W/Y, and A/G/L/M/V.
Regions of high homology are marked with solid lines and numbered. Conserved Cys residues are marked with arrowheads.
Conserved regions or amino acids that are specific for either domain 2 or domain 4 are indicated by letters.
Accumulation of mRNAs Observed in Young Zygotes Is
Dependent on Agglutination But Not on Cell Fusion
The formation of zygotes is a multistep process,
elicited by the interaction of flagella from mt1 and
mt2 gametes (Pan and Snell, 2000a). We therefore
tested which step in the mating reaction induces the
accumulation of GAS28, GAS30, and GAS31 mRNAs
seen in young zygotes. For this test, we employed
Chlamydomonas mutants that are blocked in different
steps of zygote formation. No accumulation of GAS28,
GAS30, and GAS31 mRNAs was observed in mutant
imp-5 after mixing with wild-type gametes (Fig. 5A).
Mutant imp-5 lacks mt1-specific agglutinin and thus
shows no agglutination reaction (Goodenough et al.,
1976; Ferris et al., 2005). In wild-type gametes, an
increase in GAS31 mRNA levels was observed already
30 min after mixing of competent gametes; a significant
increase in GAS28 and GAS30 mRNAs was seen only
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Hoffmann and Beck
Figure 3. Homologies of GAS28, GAS30, and GAS31 to extracellular matrix proteins of the volvocales family. Alignment of the
N-terminal domains of GAS28, GAS30, and GAS31 (domain 2 in Fig. 1B) and the N-terminal regions of three proteins encoded
by the Volvox pherophorin gene family: Pherophorin I precursor (Per1; accession no. P81131), Pherophorin II (Per2; accession
no. P81132), and Sulfated surface glycoprotein 185 precursor (SSG_185; accession no. P21997). Conserved residues are
highlighted and grouped as described in the legend of Figure 2. Lines mark conserved regions, and numbers refer to those used in
Figure 2. Conserved Cys residues are marked with arrowheads.
1 h after start of mating. The imp-3 mutant, defective in
the production of intracellular cAMP in response to the
adhesion reaction (Saito et al., 1993), showed, at most, a
very weak accumulation of GAS28, GAS30, and GAS31
mRNAs 1 h after the mixing of gametes (Fig. 5A).
We next tested whether cell fusion is a prerequisite
for induction by using two mutants shown previously
to be defective in zygote formation. Mutant fus1 is
defective in a protein named fringe that, at the tip of
the mating-type structure, is responsible for the contact with mt2 gametes (Ferris et al., 1996; Misamore
et al., 2003). In mutant ida5, the actin gene is affected
that, in mt1 gametes, leads to a defect in the assembly
of mating tubules (Kato-Minoura et al., 1997). After the
flagella-mediated adhesion reaction of wild-type gametes with either of the fusion-defective mutants fus1
or ida5 mt1, we observed mRNA accumulation for all
three GAS genes that was similar to that seen after the
mixing of wild-type gametes (Fig. 5B). These results
indicate that the agglutination reaction and an increase
in intracellular cAMP levels are required for increased
gene expression; gamete fusion, however, apparently
is not needed.
GAS28, GAS30, and GAS31 Expression in
Activated Gametes
The interaction of flagella from gametes of mating
types 1 and 2 in the early phase of mating is known to
elicit a rise in intracellular cAMP levels (Pasquale and
Goodenough, 1987; Zhang et al., 1991; Saito et al.,
1993). We therefore tested whether the accumulation
of GAS mRNAs is mediated by the agglutination reaction itself or via an increase in intracellular cAMP
levels. Here, we made use of observations showing
that gametes may be activated either by adding flagella isolated from mature gametes or by addition of
dibutyryl cAMP (db cAMP) to gametes (Snell and
Moore, 1980; Goodenough, 1986; Kurvari et al., 1995;
Pan and Snell, 2000a, 2000b). When isolated flagella
Figure 4. Changes in the amounts of GAS28, GAS30, and GAS31
mRNAs during gametogenesis and after zygote formation. At time 0,
the nitrogen source was removed from vegetatively growing cells.
Incubation was continued in continuous light. Zygotes were generated
by mixing mating-competent gametes of opposite mating types.
Samples for RNA isolation were removed at the times indicated (h).
Ten micrograms of total RNA per lane were loaded from gametes and
5 mg from zygotes. Northern-blot hybridizations were performed using
probes specific for GAS28, GAS30, and GAS31 mRNAs as outlined in
‘‘Materials and Methods.’’ FUS1 served as a control for a gene expressed during gametogenesis but turned off upon zygote formation. A
probe for HSP70B encoding a plastidic chaperone (Drzymalla et al.,
1996) served as a loading control. The percentage of gametes or
zygotes at each time point was determined as described in ‘‘Material
and Methods’’ and is given below the autoradiograms. The percentage
of zygotes was calculated from the number of quadriflagellate and
biflagellate cells (Beck and Acker, 1992).
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Wall Shedding and Osmotic Stress Induce Cell Wall Genes
(Fig. 6B). GAS31 mRNA accumulated already 40 min
after adding db cAMP. After 90 min, the mRNA
showed a further increase. GAS28 and GAS30 mRNAs
clearly have accumulated after 90 min. We conclude
that gamete activation, elicited by an increase in intracellular cAMP levels, caused either by the agglutination reaction or the exogenous addition of db cAMP,
induces the accumulation of GAS28, GAS30, and
GAS31 mRNAs.
Accumulation of GAS mRNAs after Cell Wall Removal
Figure 5. Analysis of GAS28, GAS30, and GAS31 expression in
mutants unable to form zygotes. Gametes (G) of mating type 1 from
the wild type and mutants were generated as described in ‘‘Materials
and Methods’’ and mixed for 0.5 or 1 h with wild-type gametes of
opposite mating type. Mating type 1 gametes from the wild type served
as a control. Samples were taken for RNA isolation, and northern-blot
hybridizations were performed using probes that are specific for
GAS28, GAS30, and GAS31 transcripts. FUS1 expression was used
as a molecular marker to monitor zygote formation since its mRNA
disappears in young zygotes (Ferris et al., 1996). A probe for HSP70B
served as a loading control. The agglutination reaction (Aggl.) was
monitored in a semiquantitative manner: 2, no agglutination; 111,
strong agglutination. The mating type (mt) is indicated by 1 and 2. A,
Assay of a mutant defective in mating type 1 agglutinin (imp-5) and of
a mutant deficient in the accumulation of cAMP (imp-3). B, Assay of
fusion-defective mutants fus1 and ida5, both of which are mt1. The
fus1 mutant has a deletion in the FUS1 gene, resulting in a smaller
transcript (Ferris et al., 1996).
from gametes were incubated with gametes of opposite mating type for 1 h, we observed a pronounced
increase in GAS28, GAS30, and GAS31 mRNA levels
(Fig. 6A).
To answer the question whether cAMP addition also
elicits an increase in mRNA levels, we incubated mature gametes with 10 mM db cAMP for 40 and 90 min
One cellular response caused by rising cAMP levels
in gametes is the GLE-mediated removal of the gametes’ cell wall (Buchanan et al., 1989; Pan and Snell,
2000a). To test whether the cAMP-induced cell wall
removal may cause an enhanced expression of GAS28,
GAS30, and GAS31, we incubated gametes from the
agglutinin-defective mutant imp-5 (Goodenough et al.,
1976; Ferris et al., 2005) with an autolysin-containing
supernatant from zygotes. The active component in
autolysin is a metalloprotease named GLE. The mutant imp-5 was used to avoid the activation of gametes
by agglutinins (see Figs. 5A and 6A) that might be present in the autolysin suspension. The GAS28, GAS30,
and GAS31 mRNA levels increased in a time-dependent manner after treatment with autolysin (Fig. 7A).
We next treated vegetative cells with autolysin. The
mRNA levels of GAS28, GAS30, and GAS31, which in
vegetative cells are very low, increased strongly as a
consequence of autolysin treatment (Fig. 7B). The removal of cell walls by this treatment was assured by
inspection of cells during treatment with Triton X-100.
Treatment with the detergent results in a rapid lysis of
cells lacking a wall; those with a wall are not destroyed.
Figure 6. Changes in GAS28, GAS30, and GAS31 mRNA levels after
gamete activation. Mature gametes were generated as described in
‘‘Materials and Methods.’’ The percentage of gametes is given below
the autoradiograms. Northern-blot hybridizations were performed
using probes for GAS28, GAS30, and GAS31 transcripts. CBLP encoding a Gb-like polypeptide served as a loading control. A, Wild-type
gametes of mating type plus were either not treated with isolated
flagella (2) or treated for 1 h with isolated flagella from gametes of
mating type minus (1). B, Wild-type gametes were treated with 10 mM
db cAMP for the time indicated.
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suspension is not involved in the activation of GAS28,
GAS30, and GAS31. There was also no difference in
mRNA accumulations of GAS28, GAS30, and GAS31
between wild-type cells and the flagellaless mutant
bld2, indicating that signaling via the flagella is not
involved. In summary, these results suggest that the
GLE-mediated cell wall removal elicits the signal that
activates gene expression.
Figure 7. Effect of autolysin treatment on GAS28, GAS30, and GAS31
mRNA levels in gametes and in vegetative cells. At time 0, cells were
treated with autolysin for different durations, and samples were taken
for RNA isolation at the time points indicated. Northern-blot hybridizations were performed using probes that detect the GAS28, GAS30,
and GAS31 transcripts. CBLP was used as a loading control. A,
Gametes of the agglutinin 1 defective mutant imp-5, generated as
described in ‘‘Materials and Methods.’’ B, Vegetative cells of the wild
type (CC-124) grown in TAP medium.
Was this induction caused by cell wall removal or,
possibly, by compounds present in the autolysincontaining suspension obtained from mating gametes?
This question was first approached by inactivation of
GLE with either EDTA or by heat treatment (Matsuda
et al., 1984, 1987). Vegetative cells treated with autolysin for 2 h either without EDTA or with 1 or 2 mM
EDTA were compared (Fig. 8A). The addition of 1 mM
EDTA strongly reduced the accumulation of GAS28,
GAS30, and GAS31 mRNAs. The increases in mRNA
levels were completely abolished by 2 mM EDTA.
Since EDTA may block other active ingredients
contained in the autolysin suspension, we investigated
whether the autolysin-mediated induction of GAS28,
GAS30, and GAS31 gene expression may be abolished
by heat inactivation of GLE. It was shown previously
that the activity of purified GLE is lost completely by
a treatment at 50°C for 5 min (Matsuda et al., 1984).
The autolysin suspension was incubated at 50°C for
10 min prior to addition to vegetative cells. The induction of GAS28, GAS30, and GAS31 expression was
completely abolished in cells incubated with the heatinactivated autolysin (Fig. 8B).
We also investigated whether small molecules present in the autolysin suspension might be responsible
for the induction of GAS28, GAS30, and GAS31 expression. For this purpose, we dialyzed the autolysin
suspension against Tris-acetate phosphate (TAP)
medium. Wild-type cells and flagellaless mutant bld2
(Goodenough and St. Clair, 1975) were incubated with
nondialyzed and dialyzed autolysin for 2 h, and the
gene expression patterns were compared (Fig. 8C). A
comparison between wild-type cells and the flagellaless mutant should reveal possible influences of the
flagella on the induction of the GAS genes. No differences in the expression patterns of GAS28, GAS30, and
GAS31 could be observed between cells treated with
nondialyzed and dialyzed autolysin. These results indicate that a dialyzable component in the autolysin
GAS Gene Activation in Response to Changes in
Osmotic Conditions
Cell wall removal in yeast was shown to activate
a branch of the high osmolarity glycerol MAP kinase
signaling pathway, resulting in the expression of genes
necessary for cell integrity (Alonso-Monge et al., 2001;
Reiser et al., 2003). It was postulated that activation
is caused by a reduction in turgor. To test whether
changes in osmotic conditions may induce the activation of GAS28, GAS30, and GAS31, vegetative cells
grown in TAP medium were shifted to TAP medium
containing 0.2 M sorbitol. The three genes were activated by this shift to hyperosmotic conditions although
with different kinetics (Fig. 9A). GAS31 mRNA accumulated weakly already 0.5 h after shift and reached
a maximal level about 1.5 h later. GAS28 and GAS30
mRNAs started to accumulate with a delay of about
1.5 h after shift to TAP medium containing 0.2 M
sorbitol. The dependence of this induction on the solute employed was tested by shifting vegetative cells
to hyperosmotic conditions employing different concentrations of NaCl in TAP medium. We used only
low concentrations of NaCl because cells started to die
after the addition of NaCl to a final concentration of
.0.1 M. As seen with sorbitol, a strong increase in
GAS28, GAS30, and GAS31 mRNA levels was noted
after a shift of cells to 0.01 M NaCl (Fig. 9B). The
GAS31 transcript accumulated to even higher levels in
cells cultured in medium with 0.05 M NaCl, whereas
the mRNA levels of GAS28 and GAS30 mRNA tended
to decline at the elevated NaCl concentration. These
results indicate that GAS28, GAS30, and GAS31 are
induced when cells encounter hyperosmotic conditions.
For yeast, it has been described that the cell wall
integrity pathway becomes activated as well by shifting cells to hypoosmotic conditions (Davenport et al.,
1995). To test these conditions for C. reinhardtii, cells
grown in TAP medium of 53 osmol (mOsM) were
shifted to distilled water (0 osmol). After incubation
for 2 h, GAS28, GAS30, and GAS31 mRNA levels of
water-incubated cells were compared with untreated
cells (Fig. 9C). Since the transcripts of all three GAS
genes accumulated in cells shifted to water, GAS28,
GAS30, and GAS31 expression clearly can be activated
by both hyperosmotic and hypoosmotic conditions.
The removal of the cell wall in plant cells results in
a reduction of turgor pressure, which leads to an influx
of water and cell expansion. We next wanted to find
out whether gene expression of GAS28, GAS30, and
1006
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Wall Shedding and Osmotic Stress Induce Cell Wall Genes
GAS31 observed upon cell wall removal (Fig. 7) is elicited by changes in osmotic conditions within the cell
(water influx) or by a signal that is generated by the
detachment of the cell wall from the cell membrane.
For the cytosol of C. reinhardtii, a value of 120 osmol
has been determined (Luykx et al., 1997a). Removal of
the cell wall in TAP medium (53 osmol) is expected to
change the osmotic conditions within the cells due
to water influx. Removal of the wall under isosmotic
conditions (medium with 120 osmol) is expected not to
change the intracellular osmotic conditions. We performed the autolysin-mediated cell wall removal in
medium with osmolarities of 93, 120, and 150 osmol
achieved by adding sorbitol to TAP medium. Vegetative cells were incubated for 2 h in media of different
osmotic values in the presence or absence of autolysin.
Analysis of GAS28, GAS30, and GAS31 expression levels revealed an induction of all three GAS genes after
cell wall removal not only under slightly hypoosmotic
conditions (93 osmol) but also under isoosmotic and
slightly hyperosmotic conditions (120 and 150 osmol,
Figure 8. Treatments that affect the autolysin preparations. Vegetative
cells were treated with autolysin preparations for 2 h before samples
were removed for RNA isolation and northern-blot hybridizations
performed using probes specific for the GAS28, GAS30, and GAS31
transcripts. CBLP was used as a loading control. A, Effect of autolysin in
the presence or absence of EDTA. Wild-type cells grown in TAP
medium were either treated with autolysin (1) or not (2). EDTA was
added at the same time at the final concentrations indicated. B, Effect of
autolysin that has been heat treated at 50°C for 10 min (A50).
Expression levels were compared with nontreated autolysin (A0). Cells
without autolysin treatment were used as control (2). C, Effect of
dialyzes of autolysin. Autolysin was dialyzed overnight against TAP
medium. Wild-type cells (CC-621) and flagellaless mutant bld2 (CC479) grown in TAP medium were treated with dialyzed autolysin (AD),
nondialyzed autolysin (A), or not treated (2).
Figure 9. Changes in GAS28, GAS30, and GAS31 mRNA levels after
a shift of vegetative cells to hyperosmotic and hypoosmotic conditions.
Wild-type cells grown in TAP medium under continuous light were
sedimented by centrifugation and resuspended in medium of different
osmolarities. Northern-blot hybridizations were performed using
probes that detected the GAS28, GAS30, and GAS31 transcripts.
CBLP was used as a loading control. A, Kinetics of GAS28, GAS30,
and GAS31 mRNA accumulation in cells shifted to TAP medium
containing 0.2 M sorbitol (time 0). Samples for RNA isolation were
taken at the time points indicated. B, Cells were shifted to TAP medium
containing the NaCl concentrations indicated, and probes were taken
after incubation for 2 h. C, Cells were resuspended in water and
compared with cells resuspended in TAP medium. Samples for RNA
isolation were taken after a treatment for 2 h.
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Hoffmann and Beck
respectively; Fig. 10). The degree of mRNA accumulation observed after cell wall removal was about
equal under all three osmotic conditions. These results
suggest that the autolysin-mediated activation of
GAS28, GAS30, and GAS31 is not caused by osmotic
changes within the cell but by cell wall removal.
Influence of Inhibition of Cytoplasmic Protein Synthesis
on GAS28, GAS30, and GAS31 mRNA Accumulation
To gain first indications on the signaling pathways
involved in the activation of these genes, we tested
whether, upon zygote formation, cell wall removal, or
the application of osmotic stress, and inhibition of
cytoplasmic protein synthesis would affect the accumulation of GAS28, GAS30, and GAS31 mRNAs. It
has been shown before that this inhibition does not
prevent gametes to agglutinate and to form zygotes
(Ferris and Goodenough, 1987). We treated mature
gametes of both mating types with 10 mg/mL cycloheximide (CHI) for 30 min and then mixed them for
0.5 or 1 h (Fig. 11A). GAS31 mRNA levels increased
strongly with kinetics similar to those seen in untreated gametes after fusion (see Figs. 4 and 5A). In
contrast, CHI prevented the accumulation of GAS28
and GAS30 mRNAs during zygote formation.
To analyze the influence of CHI on the mRNA levels
of these genes after autolysin treatment, vegetative
cells were incubated for 30 min with 10 mg/mL CHI
prior to autolysin treatment for 0.5 and 1.5 h. Again,
GAS31 mRNA accumulation was not prevented by
CHI; rather, an enhanced accumulation was observed
when compared to the untreated control (Fig. 11B).
CHI, however, completely inhibited the accumulation
of GAS28 and GAS30 mRNAs.
To test the influence of CHI on the expression of
GAS28, GAS30, and GAS31 upon application of osmotic stress, vegetative cells were preincubated for
30 min with 10 mg/mL CHI prior to transfer to TAP medium containing 0.15 M sorbitol. Here, too, an increase
Figure 10. Effect of autolysin treatment on GAS28, GAS30, and GAS31
mRNA levels under isosmotic conditions. Vegetative cells were treated
either with autolysin (1) or without autolysin (2) for 2 h in TAP medium
with different concentrations of sorbitol. Osmolarity of the medium is
given in osmol (mOsM). Northern-blot hybridizations were performed
using probes that detect the GAS28, GAS30, and GAS31 transcripts.
CBLP was used as a loading control.
Figure 11. Consequences of inhibition of cytoplasmatic protein synthesis on GAS28, GAS30, and GAS31 mRNA levels in cells after zygote
formation, after autolysin treatment, and after a shift to hyperosmotic
conditions. Northern-blot hybridizations were performed using probes
specific for the GAS28, GAS30, and GAS31 transcripts. CBLP served as
a loading control. A, CHI treatment of gametes and of gametes during
mating. Mature gametes of mating types 1 and 2 were generated as
described in ‘‘Materials and Methods’’ and then suspended in nitrogenfree medium containing 10 mg/mL CHI for 30 min. These gametes were
mixed for mating for 0.5 or 1 h, and samples were taken for RNA
isolation. B, Influence of CHI on GAS28, GAS30, and GAS31 mRNA
levels in vegetative cells treated with autolysin. Wild-type cells were
incubated with 10 mg/mL CHI for 30 min prior to autolysin treatment
(1) and compared to nontreated cells (2). Autolysin treatment started
at time 0, and samples for RNA isolation were taken at the time points
indicated. C, Effect of inhibition of cytoplasmatic protein synthesis on
GAS gene expression in cells shifted to hyperosmotic conditions. Wildtype cells in TAP medium were either treated with 10 mg/mL CHI for
30 min (1) or not (2) prior to the addition of sorbitol (0.15 M final
concentration). Cells were incubated for different durations (h). Sample
8* was treated for 8 h in TAP medium containing 10 mg/mL of CHI
without sorbitol. Northern-blot hybridizations were performed as described in ‘‘Materials and Methods.’’
1008
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Wall Shedding and Osmotic Stress Induce Cell Wall Genes
in GAS31 mRNA levels was not prevented by CHI (Fig.
11C). Similar to the observations made after cell wall
removal, the accumulation of GAS31 mRNA was distinctly higher in cells treated with CHI when compared
to nontreated cells. No GAS31 mRNA accumulation
was observed in cells treated with CHI in the absence of
osmotic stress, showing that CHI alone did not influence the expression of this gene. GAS28 and GAS30
mRNAs did not accumulate in cells treated with
a combination of sorbitol and CHI (Fig. 11C). Upon
prolonged incubation of cells in sorbitol (21 h) in the
absence of CHI, a reduction in GAS28, GAS30, and
GAS31 mRNA levels was observed (Fig. 11C). Microscopy examination of cells kept for 21 h in the presence
of 0.2 M sorbitol revealed that most of the cells were
dead.
The similarity in response of the three genes, irrespective of the conditions used for their induction, suggests the involvement of similar or identical signaling
pathways. However, the accumulation of GAS31 transcripts in the presence of CHI and the absence of
GAS28 and GAS30 mRNAs under these conditions
may indicate a divergence of these pathways at a later
step.
genes controlled by this program are GAS28, GAS30,
and GAS31, which are up-regulated in the late phase of
gamete formation, i.e. when mating competent cells
already are formed (Fig. 4). Since the gene products
appear to be members of the HRGP family of cell wall
proteins, a function of these proteins in mature and
nondividing gametes is difficult to discern, and we
previously speculated that these mRNAs, produced
in gametes, may by stored for later translation in
the newly formed zygotes (Rodriguez et al., 1999). Preliminary results from studies with GAS31-specific antibodies indicate the presence of this protein in young
zygotes, while in gametes or vegetative cells, only a
weak signal was detectable (our unpublished data).
The mating of gametes elicited a second increase
in the mRNA levels of these GAS genes (Fig. 4). The
shedding of the gametes’ wall appears to be the ultimate cue that is responsible for their induction (Fig.
12). Since gametes, after shedding of their walls, fuse
within minutes after mating (Harris, 1989), these
mRNAs, present in gametes and accumulating with
DISCUSSION
The gene products of GAS28, GAS30, and GAS31
exhibit the typical feature of C. reinhardtii cell wall
proteins: a ser(pro)x-rich domain. Such protein domains
are subject to glycosylation and may form fibrous
structures (Woessner and Goodenough, 1992; Ferris
et al., 2001, 2005). The predicted fibrous structures of
the three GAS proteins are flanked by domains that
were predicted to form globular structures. Proteins of
this type have been classified as chimeric extensins
(Kieliszewski and Lamport, 1994). Although GAS28,
GAS30, and GAS31 do not exhibit homology to known
C. reinhardtii cell wall proteins, they share the structural features of globular heads and Hyp-rich shafts
with the majority of HRGPs from this organism (Ferris
et al., 2001).
The presence of four additional genes that may
encode proteins with homology to GAS28 and GAS30
was revealed by a search for related genes in draft
version 2 of the C. reinhardtii genome (Fig. 2). These results indicate that GAS28 and GAS30 are members of
a larger protein family. The N-terminal domains of
GAS28 and GAS30 also share conserved sequence
motifs with GAS31 and pherophorins I and II as well
as SG185, proteins of the extracellular matrix of Volvox
(Fig. 3). This supports the hypothesis that different
domains of HRGPs are conserved within the order of
volvocales and might have evolved from a common
ancestor (Woessner et al., 1994).
Successful mating and the subsequent formation of
zygotes in C. reinhardtii are based on a program of
differentiation involving the activation and inactivation of genes in a predetermined sequence. Among the
Figure 12. Model for the control of GAS28, GAS30, and GAS31
expression by endogenous and extrinsic signals. The adhesion reaction
between mature gametes elicits an increase in intracellular cAMP
levels, which in turn causes the activation of GLE. GLE catalyzes the
degradation of the cell wall, which is proposed to lead to a signal that
induces GAS28, GAS30, and GAS31.
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Hoffmann and Beck
accelerated rates in young zygotes, may serve as template for the rapid synthesis of proteins needed for the
regeneration of a new wall protecting the young zygotes. While we speculate that these mRNA accumulations are a consequence of increased transcription of
the genes, an alternative explanation is a modulation
of RNA levels by changes in their stability. These two
alternative routes to increases in mRNA levels have
not yet been elucidated.
This induction of the three GAS genes parallels
previous findings in which a screening for genes expressed in activated gametes resulted in the identification of a cDNA named G47. The gene for G47 encodes
a putative cell wall protein; its mRNA accumulated in
gametes upon activation and also in dewalled vegetative cells. Whether its induction in activated gametes is
linked to cell wall shedding has not been analyzed but,
in view of the results presented here, appears to be
possible. In summary, these data point to the important role played by the gametes’ cell wall shedding in
the generation of a signal that controls the expression
of genes during the mating reaction.
Vegetative cells also were shown to be competent for
the activation of GAS28, GAS30, and GAS31 by cell
wall removal (Fig. 7B), suggesting that the signal is not
restricted to cells undergoing gametogenesis. This idea
is supported for GAS28, since the gene for this protein
is up-regulated after mitosis in dividing cells when
new cell walls are being elaborated by the daughter
cells (Abe et al., 2004). That alterations, affecting the
wall of vegetative cells, generate a signal resulting in
the activation of cell wall genes has been demonstrated
previously. Three genes, GP1, GP2, and Mrp47, encoding proteins of the HRGP family, in vegetative cells are
induced by autolysin treatment (Adair and Apt, 1990;
Kurvari, 1997). Since they were not discovered in
screens designed to detect genes expressed in young
zygotes, they likely are specific for vegetative cells and
gametes and thus similar to VSP-3, another cell wall
gene induced by removal of the wall that is not expressed in young zygotes (Woessner et al., 1994). Increased expression of genes after cell wall removal
possibly may be caused by a general stress response.
This, however, appears to be unlikely since the application of heat stress did not induce GAS28 (von
Gromoff and Beck, 1993) and GAS30 (data not shown).
How is the activation of the three GAS genes elicited
by wall removal? The intracellular osmotic status is
expected to be affected by removal of the wall when
performed in TAP medium since this medium with 53
osmol has a lower osmotic strength than that of the C.
reinhardtii interior, which is isosmotic with a medium
of 120 osmol (Luykx et al., 1997a). Cell wall removal is
expected to lead to higher activity of the contractile
vacuole and/or cell expansion through water influx,
possibly causing the activation of osmosensors and
linked signaling pathways. However, wall shedding
under isosmotic conditions also elicited an induction
of the GAS genes (Fig. 10). Thus, the signal appears to
be generated by detachment of the cell wall from the
plasma membrane. Here, Saccharomyces cerevisiae
may provide a model where an activation of the
MAP-kinase-mediated high osmolarity glycerol pathway in response to wall removal (seen even in cells
adapted to hyperosmotic conditions) is registered by
trans-membrane osmosensory His kinase SLN1 possibly via an ectodomain that may form contacts between
the plasma membrane and the cell wall (Hohmann,
2002; Reiser et al., 2003). A relationship between the
osmo-sensing system in yeast and that in higher plants
was deduced from the observation that Arabidopsis
(Arabidopsis thaliana) cytokinin response 1 (Cre1) protein, also a His kinase, could complement the defective
response to cell wall removal of yeast mutants lacking
SLN1 (Reiser et al., 2003). A gene for a His kinase with
homology to SLN1 and Cre1 also is present within the
C. reinhardtii genome (gene model C_930062). In view
of the gene activation observed under isosmotic conditions, also in C. reinhardtii, loss of contact between
the plasma membrane and the cell wall is postulated to
trigger a signaling pathway that results in the induction of the three GAS genes (Fig. 12). This pathway also
may be employed when cells are shifted to hyperosmotic conditions, since shrinking of the cytoplasmic
membrane involves its partial detachment from the wall.
The three GAS genes analyzed are also induced
by exposing the algal cells to hyperosmotic or hypoosmotic conditions. The addition of either sorbitol or
NaCl or a shift of cells from TAP medium to water elicited a pronounced activation of the three genes (Fig. 9).
These genes, to our knowledge, are the first described
in C. reinhardtii for which an osmoregulation is demonstrated. The osmosensors involved as well as the
participating signaling pathways remain to be elucidated as well as the question whether signaling components employed here are the same as those used by
the pathway activated by cell wall removal. One argument for the participation of distinct pathways comes
from differences in the kinetics of RNA accumulation.
Thus, GAS28 and GAS30 mRNA clearly have accumulated 1 h after cell wall removal (Fig. 7B), while this
accumulation is delayed upon application of hyperosmotic conditions (Fig. 9A). In contrast, GAS31 responded with similar RNA accumulation kinetics
to both sets of conditions. Besides these differences,
GAS31 regulation is distinguishable from that of
GAS28 and GAS30, since its induction, but not that of
the latter genes, under all conditions tested (wall removal and hypoosmotic or hyperosmotic conditions)
occurred in the absence of de novo protein synthesis
(Fig. 11). This implies that the accumulation of GAS28
and GAS30 mRNAs is dependent on the de novo
synthesis of protein factors (Fig. 12). These data are suggestive evidence for the existence of different osmoregulated signaling pathways in Chlamydomonas.
Whether the situation is as complex as in S. cerevisiae,
where multiple osmosensors and coupled signaling
pathways participate in the regulation of gene expression in response to hypoosmotic and hyperosmotic
conditions (Hohmann, 2002), remains to be elucidated.
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Wall Shedding and Osmotic Stress Induce Cell Wall Genes
Another signal that during mating and zygote
formation triggers the activation of genes is gamete
fusion. Several genes that are expressed after cell
fusion (zygote-specific genes) do not show activation
in mutants defective in gamete fusion (Ferris and
Goodenough, 1987). Two of these zygote-specific genes
encode components of the cell wall (Woessner and
Goodenough, 1989; Matters and Goodenough, 1992).
While additional genes have been classified as zygote
specific, their activation by cell fusion has not been
demonstrated in a rigorous manner (Uchida et al.,
1993). These latter genes may possibly be induced by
a signal generated prior to fertilization, i.e. gamete
activation and cell wall shedding.
Altered gene expression in response to cell wall
alterations and osmotic conditions is a topic of importance in plant physiology (Bray et al., 2000). Osmoregulation in higher plants is due to complex interactions
of regulatory metabolites at different levels of organization: organs, tissues, and cells. The basics of this
regulation thus are easier to study in unicellular organisms. The characterization of three genes in C. reinhardtii
that respond to these cues opens up the route to study
the signaling processes involved in this simple plant.
Additional attractive aspects are the availability of osmoregulatory mutants (Luykx et al., 1997b) and strains
that completely lack a cell wall but are viable even
when suspended in water.
MATERIALS AND METHODS
Strains and Cell Culture
We used seven Chlamydomonas reinhardtii strains in this study: wild-type
strains CC-620 (mt1) and CC-124 (mt2), the agglutinin 1 defective mutant
imp-5 (CC-469; Adair et al., 1983), a mutant deficient in the accumulation of
cAMP (imp-3, CC-465; Goodenough et al., 1976), the fusion-defective mutant
fus1 (CC-1158; Ferris et al., 1996), and the flagellarless mutant bld2 (Goodenough and St. Clair, 1975), all obtained from the Chlamydomonas Genetics
Center (Duke University, Durham, NC). The fusion-defective mutant ida5
(mt1) was kindly provided by R. Kamiya (Kato-Minoura et al., 1997).
Vegetative cells were grown photomixotrophically at 23°C in TAP medium
(Gorman and Levine, 1965) at 23°C on a shaker with continuous irradiation by
white light (fluence rate 60 mmol m22 s21) to a density of approximately 6 3
106 cells per mL. Fluorescent light (Osram L36W/25) was used for illumination. For induction of gametogenesis, vegetative C. reinhardtii cultures at
a density of approximately 5 3 106 cells/mL were concentrated by centrifugation (3000g for 5 min). TAP medium was carefully removed, and the cells
were resuspended in nitrogen-free TAP medium at a density of about 5 3 106
cells/mL. Subsequently, cells were incubated with shaking for 5 to 17 h.
Mating and determination of the percentage of gametes were performed as
described previously (Treier et al., 1989; Beck and Acker, 1992).
Protein synthesis inhibition was performed by incubating cells for 30 min
with 10 mg/mL of CHI (obtained from Sigma-Aldrich) prior to additional
experimental treatments.
Activation of Gametes
Gametes were activated for mating either by incubation with flagella from
gametes of opposite mating type or by the addition of db cAMP. Flagella were
isolated from mature gametes (1 L culture, 64% mating) as described by Pan
and Snell (2000b). Flagella were resuspended in 500 mL resuspension buffer
(20 mM HEPES, pH 7.2, 5 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol,
containing a 1/100 dilution of the Sigma-Aldrich protease inhibitor mixture
for plant cells). Flagella suspension (100 mL) was added to 15 mL of mature
gametes and incubated for 1 h prior to harvest on ice. Gametes were also
activated by incubating them with 10 mM db cAMP (Fluka) and freshly prepared 0.15 mM paperverine (Sigma-Aldrich) for different durations (Wilson
et al., 1997).
Removal of Cell Walls
Crude autolysin preparations were obtained by mixing mature gametes (13
107 cells per mL) of opposite mating types and incubating them for 1 h in the
dark without shaking. The percentage of zygotes in the mixture was estimated
by counting the biflagellated and quadriflagellated (zygotes) cells after
fixation in 1% glutaraldehyde. When .80% of the gametes have mated, the
autolysin-containing supernatant was collected by spinning down the cells at
2000g at 4°C for 5 min. The supernatant was recentrifuged at 13000g for 10 min
at 4°C. The autolysin-containing supernatant was sterilized by filtration using
syringe filters (Roth) and frozen in aliquots. For removal of the cell walls, 1 mL
of autolysin was added to 10 mL of cells (density 63106 cells per mL). To
monitor the removal of the cell walls, 10 mL of treated cells were incubated
with 10 mL of 1% Triton X-100 for 5 min. Untreated cells served as a control.
Cells were counted under a microscope and compared to cells not treated with
Triton X-100. Cells without a cell wall lyse in the presence of Triton X-100.
Osmotic Stress Conditions
For hyperosmotic stress, cells were grown in TAP for 3 d to a density of
6 3 106 cells/mL, harvested by centrifugation, and resuspended in TAP
containing a final concentration of 0.15 or 0.2 M sorbitol or a final concentration
of 0.01 or 0.05 M NaCl. For hypoosmotic stress, cells were resuspended in
destilled water. Osmolarity of the medium was determined by measuring the
media used for culturing with an osmometer (OM 801 from Vogel).
RNA Isolation and Northern Blots
For RNA isolation, cells were harvested by centrifugation for 5 min at 4°C,
resuspended in 23 lysis buffer (0.6 M NaCl, 10 mM EDTA, 100 mM Tris-HCl,
pH 8.0, and 4% SDS), and then frozen in liquid nitrogen. The lysates were
extracted with phenol (Sambrook et al., 1989). The RNA was precipitated with
0.33 volume of 8 M LiCl at 4°C overnight (Barlow et al., 1963). The pellet was
suspended in diethylpyrocarbonate-treated water, mixed with 0.1 volume of
3 M sodium acetate (pH 6.0), and precipitated with 2.5 volumes of ethanol at
220°C. Total RNA (10 mg/lane) was fractionated on 1.2% (w/v) agarose/
formaldehyde gels, transferred to Hybond-N1 membranes (Amersham), using
25 mM sodium phosphate buffer (pH 6.5), and baked at 80°C for 2 h. The
probes used for hybridization were as follows: GAS28 (a NheI-XhoI fragment
of 690 bp from the 3# end of GAS28 cDNA), GAS30 (a NruI-XhoI fragment of
1000 bp from the 3# end of the genomic DNA), GAS31 (a XbaI-XhoI fragment of
1050 bp from the 5# end of the genomic DNA), FUS1 (an EcoRV-BamHI
fragment of 1700 bp from plasmid imp1-17; Ferris et al., 1996), HSP70B (von
Gromoff et al., 1989), and CBLP, which encodes a Gb-like polypeptide (von
Kampen et al., 1994). Probes were labeled with 50 mCi [a-32P]dCTP
(Amersham) according to the random priming protocol (Feinberg and
Vogelstein, 1983). Hybridizations were performed as described previously
(Wegener and Beck, 1991). After hybridization, the membranes were washed
twice in 23 SSC for 5 min at room temperature, once in 23 SSC, 1% SDS for 30
min at 65°C, and once in 0.13 SSC, 1% SDS for 30 min at 65°C.
Isolation of the GAS28, GAS30, and GAS31 Genes
GAS28 and GAS31 were cloned by screening the membrane-immobilized
DNA of a C. reinhardtii BAC library (Lefebvre and Silflow, 1999) obtained from
the Clemson University Genomics Institute (Clemson, SC, http://www.
genome.clemson.edu/) using a NheI-XhoI fragment from GAS28 cDNA and
a SalI-BglII fragment from GAS29 cDNA as probes, respectively. Hybridizations of the membranes were performed using the protocol from Clemson
University Genomics Institute. Seven clones were hybridized with the GAS28
probe (21H23, 21K10, 05O05, 29O09, 30L05, 36O14, and 07G16) and three
clones with the GAS31 probe (06K23, 22G06, and 34C08). Positive BAC clones
(21H23 and 06K23) were obtained from Clemson University Genomics Institute. DNA was isolated using the protocol of the BAC clone providers
(http://cbs.umn.edu/;amundsen/chlamy/methods/bac_med.html). Genes
were subcloned in pBluescript SK1/2 (Stratagene). GAS31 cDNA clones and
Plant Physiol. Vol. 139, 2005
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Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Hoffmann and Beck
GAS29 cDNA clones were obtained by screening a l-zap cDNA library
prepared from mt1 gametes (Kurvari et al., 1995) using the 5# end of GAS31
(XbaI-XhoI fragment from the genomic subclone) and the 3# end of GAS29
cDNA (SalI-BglII fragment), respectively, as probes. Inserts of positive clones
were excised as pBluescript II SK1/2 plasmids using the Stratagene rapid
excision kit. The genomic structures of GAS28 and GAS31 were determined by
comparison of the cDNA sequences with the genomic sequences.
GAS30 was cloned by screening a cosmid library (Purton and Rochaix,
1994) using an NdeI-XhoI fragment of GAS28 cDNA as a probe. Hybridization
was performed as described by Sambrook et al. (1989). The gene was cloned
into pBluescript SK1/2. The genomic structure of GAS30 was obtained using
information from RT-PCR and 3# rapid amplification of cDNA ends (RACE)
experiments. The first-strand cDNA synthesis for the RT-PCR and 3# RACE
experiments was performed with total RNA from young zygotes using
SuperScript III reverse transcriptase (Invitrogen) and an oligo(dT)25 primer.
The reaction was performed according to the manual provided. Two microliters of the product were used for PCR reactions to amplify (1) the 5# UTR
(primer pair 5#-GCTCTTCCCTAGCCACCTTAGTAGAC-3# and 5#-GACTGGATTGAGACCT CATGCTTGC-3#) with an annealing temperature of 60°C,
(2) the coding region on both sides of the second intron (primer pair
5#-CAAGCATGAGGTCTCAATCCAGT-3# and 5#-CCACTTCTTGCCGTCCACAGTCACGTTGC-3#) with an annealing temperature of 62°C, and (3) the
3# end by a 3# RACE [primer pair 5#-CTCTAAGTGCCTCCTGATGAG-3# and
oligo(dT)25] with an annealing temperature of 55°C. Reactions were performed in a volume of 50 mL with 13 buffer (500 mM KCl, 100 mM Tris-HCl
[pH 8.8], 15 mM MgCl2, 1% Triton X-100, 0.1% gelatin), 10 mM dNTPs-mix, and
2 units Taq polymerase (Amersham). The resulting PCR products were cloned
and sequenced.
The coding regions were translated into amino acid sequences using BCM
six frame translation utilities at http://searchlauncher.bcm.tmc.edu/seq-util/
seq-util.html, and signal sequences were predicted with TargetP (Nielsen et al.,
1997; Emanuelsson et al., 2000). Secondary structures of deduced gene
products were predicted using the algorithm HNN (Guermeur et al., 1999).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers DQ017908 (GAS28), DQ017907 (GAS30),
and DQ017909 (GAS31).
ACKNOWLEDGMENTS
We wish to thank Dr. Saul Purton for intellectual input and Dr. Michael
Schroda for intellectual input and critical reading of the manuscript.
Received May 10, 2005; revised July 5, 2005; accepted July 15, 2005; published
September 23, 2005.
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