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First published online 13 March 2008
doi: 10.1242/dev.016303
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1 UPMC Université Paris 06, The Marine Plants and Biomolecules
Laboratory, UMR 7139, Station Biologique de Roscoff, Place Georges Teissier,
BP74, 29682 Roscoff Cedex, France.
2 CNRS, UMR 7139, Laboratoire International Associé Dispersal and
Adaptation in Marine Species, Station Biologique de Roscoff, Place Georges
Teissier, BP74, 29682 Roscoff Cedex, France.
3 Microarray Platform, OUEST-Génopole, Université de Rennes
1-Faculté de Médecine, Campus de Villejean, 35043 RENNES
Cédex, France.
4 Computer and Genomics resource Centre, FR 2424, Station Biologique de Roscoff,
Place Georges Teissier, BP74, 29682 Roscoff Cedex, France.
Author for correspondence (e-mail:
cock{at}sb-roscoff.fr)
Accepted 31 January 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Brown algae, Ectocarpus siliculosus, Immediate upright, Initial-cell division, Life cycle, Phaeophyceae
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Brown algae develop from cells that are released into the
surrounding seawater (de Reviers,
2002), and the early stages of development are therefore easily
observable. The growth of the initial cell can be markedly different in
different species, involving either unipolar or bipolar germination (emergence
of either one or two germ tubes, respectively). Moreover, bipolar germination
can result in different growth patterns. When germination produces a symmetric
filament, this leads to the development of a prostrate, basal structure before
the erect thallus is formed (termed mediate differentiation or heterotrichy,
see Table 1). By contrast, if
an asymmetric structure is formed following germination, this leads to the
immediate development of an erect thallus without the formation of a
prostrate, basal structure (immediate differentiation)
(Pedersen, 1984;
Fletcher, 1987).
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;
Scheres and Benfey, 1999).
Similarly, a symmetric division is defined as one that generates daughter
cells that are destined to acquire the same developmental fate
(Morrison and Kimble, 2006).
Zygotic/initial cell divisions frequently elaborate polarity that underlies a
major body axis [Berleth and Chatfield,
2002
(http://www.aspb.org/publications/arabidopsis/);
Schneider and Bowerman, 2003;
Huynh and St Johnston, 2004].
Within the brown algae, efforts to dissect the processes of polarisation and
the first cell division have concentrated on the Fucales, where it has been
shown that polarity can be established in response to a range of external
vectors with unidirectional light being the strongest stimulus
(Quatrano, 1997).
The filamentous brown alga Ectocarpus siliculosus (Dillwyn)
Lyngbye has been proposed as a general model for the brown algae
(Peters et al., 2004a) and its
genome has been sequenced at Genoscope
(http://www.genoscope.cns.fr/spip/Ectocarpus-siliculosus,740.html).
The life cycle of E. siliculosus involves an alternation between two
macroscopic generations that differ morphologically (sporophytes produce few
laterals and develop from a branched prostrate base, whereas gametophytes are
more richly branched and devoid of a prostrate base)
(Kornmann, 1956;
Müller, 1964). In this
study, we show that, unlike the sporophyte, which is formed by mediate
differentiation following bipolar germination and symmetric division of the
initial cell (Peters et al.,
2004b), the E. siliculosus gametophyte exhibits an
asymmetric initial cell division and immediate differentiation of an erect
thallus. Therefore, the alternation of generations in E. siliculosus
involves an alternation between two fundamentally different patterns of
initial cell division: symmetric and asymmetric.
A spontaneous mutant, immediate upright (imm), exhibited several phenotypic traits characteristic of the gametophyte generation during the sporophyte generation of its life cycle, including asymmetric initial cell division. This mutant produced functional meiospores, demonstrating that symmetric initial cell division is not essential for an individual to become a functional sporophyte. However, specific alterations in the expression of generation-specific genes were detected in this mutant using a microarray approach, indicating that imm is a bona fide life cycle mutant and hence that the phenotypic traits that are modified in imm are normally under life cycle control in wild-type algae.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), autoclaved seawater. For the study of
germination, spores or gametes were allowed to settle on cover-slips. Zygotes
were produced and isolated as described by Peters et al.
(Peters et al., 2004b). The
sex of gametophytes was determined by microscopic observation of zygote
formation in hanging-drop preparations
(Kawai et al., 2005) in which
the strains to be tested had been combined with fertile thalli of male and
female reference strains. Genetic analysis of meiotic offspring followed
Müller (Müller,
1991).
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Microarray analysis of gene expression
A microarray was constructed by spotting PCR amplified inserts from two
suppression subtraction hybridisation (SSH) libraries, enriched for genes
expressed preferentially during the sporophyte and gametophyte generations,
respectively, onto glass slides. The SSH libraries were produced in a
differential screen between the partheno-sporophyte and gametophyte
generations of the life cycle using the PCR Select kit (Clontech, Mountain
View, CA, USA). Oligonucleotides corresponding to flanking vector sequences
were used to amplify inserts from 600 clones randomly chosen from each library
and these 1200 PCR products were spotted in triplicate on each microarray. RNA
was extracted as described by Apt et al.
(Apt et al., 1995) and its
quality was monitored on a Bioanalyser using an RNA 6000 Nano Assay kit
(Agilent, Santa Clara, CA, USA). Labelled cDNA targets were synthesised from
15 µg of total RNA with an oligo(dT) primer and by indirect labelling using
the CyScribe cDNA Post Labelling kit (Amersham, Piscataway, NJ, USA).
Hybridised microarrays were scanned with a GenePix 4000 (Molecular Devices
Corporation, Downingtown, PA, USA) and analysed using GenePix Pro 5.1. All
hybridisations were carried out in triplicate. Data corresponding to a set of
132 probes that did not show differential expression during the sporophyte and
gametophyte generations were used to normalise the data obtained from the
different hybridisation experiments. The average value for the coefficient of
variation was 0.29 for the wild-type partheno-sporophyte samples and 0.35 for
the imm mutant partheno-sporophyte samples. Full descriptions of the
array design, probe sequences, RNA extraction, cDNA labelling, hybridisation
and normalisation protocols are available via the ArrayExpress database
(accession number E-MEXP-848). Statistical analysis was carried out using the
Statistical Analysis of Microarrays (SAM) method
(Tusher et al., 2001) in the
TIGR MeV package, version 3.1. The cDNA fragments corresponding to probes that
had been shown to be significantly, differentially expressed by this analysis
were sequenced and compared with the EST and genomic sequence data available
for Ectocarpus siliculosus.
Quantitative PCR
Total RNA was extracted from diploid sporophytes, partheno-sporophytes and
gametophytes of both wild-type and imm strains using the Plant RNeasy
extraction kit (Qiagen, Courtaboeuf, France), and treated with RNAse-free
DNAse-I according to the manufacturer's instructions (Qiagen). The
concentration and quality of the RNA was determined by spectrophotometry and
agarose gel electrophoresis. Two milligrams of total RNA were
reverse-transcribed using the Superscript II RT kit (Life Technologies,
Gaithersburg, MD, USA) according to the manufacturer's instructions.
Oligonucleotide sequences were designed for each gene using both Primer
Express TM1.0 (PE Applied Biosystems, Foster City, CA, USA) and Oligo 4.0
(Rychlik and Rhoads, 1989).
cDNAs were amplified using the IQ Sybrgreen supermix (Biorad Laboratories,
Hercules, CA, USA) on a Chromo4 System thermocycler (BioRad Laboratories). The
amplification efficiency was tested using a genomic dilution series and was
always at least 80%. The specificity of amplification was checked with a
dissociation curve. Details of the oligonucleotides used can be provided on
request. E. siliculosus genomic DNA was used as a quantification
reference. A dilution series ranging from 37 to 48671 copies of the E.
siliculosus genome was prepared and tested for each gene amplification.
The EF1
gene was chosen as a constitutively expressed control
based on tests carried out on three Ectocarpus genes (encoding actin,
EF1 and -tubulin). Normalisation was carried out using the
EF1 data and the geNorm method (Vandesompele et al., 2003). The
normalised data were expressed as the mean±s.d. calculated from three
independent biological experiments.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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E. siliculosus sporophytes can be produced in a number of
different ways (Fig. 1): (1)
via gamete fusion and zygote production; (2) by mito-spores produced by the
plurilocular sporangia of sporophytes; (3) from a minority of the meio-spores
produced by unilocular sporangia (the majority producing gametophytes); or (4)
by parthenogenesis from settled unfertilised gametes (referred to as
partheno-sporophytes). The early development of sporophytes produced by each
of these alternative pathways was compared with the early development of the
gametophyte generation of the same strain, described above. All sporophytes
showed the same pattern of development, except that the growth of
partheno-sporophytes was slower during the first few days. Germination was
bipolar, as in the gametophyte. However, in contrast to the situation observed
in the gametophyte, the two daughter cells of the sporophyte initial cell
exhibited symmetric cell fates, producing the two ends of a symmetric
prostrate filament (Fig. 3A-E).
Because of these identical cell fates, the initial cell division in the
sporophyte is defined as symmetric despite the morphological asymmetry of the
initial cell at the time of cell division
(Fig. 3B). This follows the
definition given by Morrison and Kimble
(Morrison and Kimble,
2006).
The cells of the prostrate filament became rounder and their cell walls thickened as they became older (i.e. in the central region of the filament; Fig. 3E,F). Laterals with the same morphology as the initial filament were produced from the rounded cells, and grew along the surface of the substratum (Fig. 3F) or up into the medium. Upright filaments with cylindrical cells (20-30 µm diameter) developed on the prostrate base after 4-5 weeks to produce an erect thallus (Fig. 3G,H). The upright filaments usually emerged from older regions of the prostrate base. The mature sporophyte consisted of a well-developed, prostrate basal system from which emerged the upright filaments (Fig. 3I). Compared with gametophyte germlings, the prostrate sporophyte thalli were not easily detached from either polystyrene or glass surfaces. Plurilocular sporangia developed on upright filaments after 7 weeks, in terminal or lateral positions (Fig. 3J). Occasionally, plurilocular sporangia developed on the prostrate structure, in some cases even before the emergence of upright filaments (not shown). Unilocular sporangia formed only on upright filaments, in terminal or lateral positions, a week after the plurilocular sporangia (Fig. 3K). The upright filaments were branched (not shown), although to a lesser extent than those of a gametophyte, and, in older thalli, also produced rhizoids (not shown). Rhizoids originated only from the upright filaments and were not produced by the filaments of the prostrate structure (Fig. 3H). Overall, the sporophyte exhibited mediate differentiation because a prostrate, basal structure was formed before the development of the erect thallus (i.e. the upright filaments).
In summary, observation of the early development of the two generations of the E. siliculosus life cycle showed that they exhibited markedly different patterns of development. These two patterns of development involved either asymmetric or symmetric divisions of the initial cells of gametophytes or sporophytes, respectively. The gametophyte formed two types of vegetative structures (rhizoids and upright filaments) and one kind of reproductive structure (plurilocular gametangia), the sporophyte three types of vegetative structures (prostrate filaments, rhizoids and upright filaments) and two kinds of reproductive structure (plurilocular and unilocular sporangia).
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; Robinson and
Miller, 1997; Fowler et al.,
2004; Brownlee and Bouget,
1998). In this group, photopolarisation causes the zygote to
germinate in a negatively phototropic manner, emitting a rhizoid on the side
opposite the light source. The markedly different germination patterns of the
E. siliculosus sporophyte and gametophyte led us to test their
responses to unidirectional light. When grown in unidirectional light, the
majority of individuals of both the sporophyte and gametophyte generations
germinated in a negatively phototropic manner
(Fig. 4). However, this
response was significantly less marked in the sporophyte generation, where
only 73% of individuals germinated away from the light, compared with 93% for
the gametophytes (
2=25.68, P<0.001).
The sporophyte of the imm mutant exhibits a gametophyte-like pattern of early development
The ability of E. siliculosus to produce partheno-sporophytes from
unfertilised, haploid gametes allows the phenotypic effects of mutations to be
detected directly in both the gametophyte and the (partheno-)sporophyte
generations. Phenotypic analysis of partheno-sporophytes derived from the 120
gametophytes produced by the Ec 17 sporophyte identified one gametophyte
strain (Ec 137) that produced partheno-sporophytes with an aberrant
germination pattern. The partheno-sporophytes of this strain exhibited
asymmetric division of the initial cell rather than the usual pattern of
symmetric division of the initial cell (exhibited by the partheno-sporophytes
derived from its sibling gametophytes). The same aberrant developmental
phenotype was seen in diploid sporophytes homozygous for the mutant locus (see
below for the production of such thalli). The aberrant germination pattern
involved the production of a first germ tube that developed into a thin (3-5
µm diameter) filament that resembled the rhizoid produced by the
germinating gametophyte (compare Fig.
5A with Fig. 2A).
The second germ tube developed into an upright filament (initially 10 µm in
diameter, but widening to 20-30 µm in the upper part of the filament),
again similar to that produced by germinating gametophytes (compare
Fig. 5B with
Fig. 2D). Based on this
phenotype, the mutant was named immediate upright (imm). The
aberrant germination pattern was observed for all developing gametes of Ec 137
and was not modified by cultivation at 5, 10 or 20°C.
The aberrant early development of the imm mutant had a significant effect on the morphology of the alga later in development. After 6 weeks growth, the imm mutant had produced a well-developed erect thallus attached to the substratum with rhizoids (Fig. 5C), whereas wild-type sporophytes had, after the same period of growth, developed a dense, prostrate, basal system from which only the first upright filaments emerged (Fig. 3I). This morphological difference between wild-type and imm sporophytes was already visible under the light microscope after 1-2 weeks growth and the two types of growth habit could be distinguished with the naked eye after 4-5 weeks. This is illustrated by the macroscopic views shown in Fig. 5D,E.
Despite the resemblance of the imm partheno-sporophyte to the wild-type gametophyte, further analysis showed that it retained its sporophyte identity. Mature imm partheno-sporophytes produced not only plurilocular but also unilocular sporangia, reproductive structures that are only produced by the sporophyte generation (Fig. 5F,G). Both of these structures can either emerge directly from a filament or be borne on a short branch, in both the wild type and in the imm mutant. Mating combinations with gametophyte reference strains showed that the spores released from the plurilocular sporangia were indeed spores and not gametes (they were incapable of fusing with fertile gametes of either sex). Even during early development, and despite their resemblance to the gametophyte, imm partheno-sporophytes exhibited several features that were typical of the wild-type sporophyte. For example, imm partheno-sporophytes adhered firmly to the substratum and were difficult to detach. Moreover, when grown in unidirectional light, the negative phototropic response was less marked than that detected normally for the gametophyte and more similar to that of the wild-type sporophyte, with only 68% of the individuals tested germinating away from the light (Fig. 4). It was also noted that, although the first germ tube developed as a filament in the same manner as in the gametophyte, it was less wavy in appearance (compare Fig. 5B with Fig. 2C,D).
The meio-spores produced in the unilocular sporangia of the imm partheno-sporophytes developed into phenotypically normal gametophytes that produced functional gametes (data not shown). All asexually produced sporophytes, derived either from mito-spores produced in the plurilocular sporangia of the imm partheno-sporophytes or via the gametophyte generation by parthenogenetic germination of gametes, exhibited the imm phenotype.
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imm behaves as a stable recessive single-locus Mendelian allele
The imm mutant (strain Ec 137) was crossed with a sister
gametophyte (Ec 25) whose gametes showed a wild-type pattern of
parthenogenetic development (see Fig.
6 for a summary of the crosses carried out for the genetic
analysis). Three zygotes were raised from this cross and the resulting
sporophytes all showed a wild-type pattern of early development. The
development of one of these sporophytes (strain Ec 372) was followed to
maturity and no differences were observed compared with the developmental
pattern of a wild-type sporophyte (data not shown). This indicated that the
imm mutation was recessive and was complemented by the wild-type
allele in the diploid sporophyte.
To analyse the segregation of the imm locus in subsequent
generations, 15 unilocular sporangia were isolated individually from the
diploid sporophyte Ec 372. These sporangia, each of which contained more than
100 meio-spores (derived from a single meiosis followed by at least 5 mitotic
divisions), produced 15 `families' of gametophytes, which again exhibited a
germination pattern typical of wild-type gametophytes. For each of these
families, between 15 and 31 gametophytes were sub-isolated and their sex and
the germination pattern of their gametes (i.e. the partheno-sporophyte
generation) were recorded (see Table S1 in the supplementary material).
Overall, 155 gametophytes produced partheno-sporophytes with a wild-type
germination pattern and 191 produced partheno-sporophytes with the
imm germination pattern. These figures are consistent with a 1:1
segregation ratio and Mendelian inheritance of a single-locus recessive
mutation (2=3.746, the deviation from 1:1 has a probability
greater than 0.05).
Several additional crosses were performed to analyse further the inheritance of the imm mutation. Using the gametophytes derived from the diploid sporophyte Ec 372, crosses were performed with all possible combinations of wild-type and imm mutant gametes (Fig. 6). Eleven diploid sporophytes, homozygous for imm, were raised from the cross Ec 419 (imm) x Ec 420 (imm). Without exception, they showed the imm germination pattern, indicating full penetrance of the imm mutation in this diploid context (provided the dominant, wild-type IMM allele is absent). From one of these sporophytes (Ec 428), we isolated five unilocular sporangia. From each of the families of gametophytes that developed from them, we selected six gametophytes and recorded their sex and the germination pattern of their gametes. Regardless of their sex, all of these gametophytes produced partheno-sporophytes with the imm germination pattern.
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Finally, from the reciprocal crosses Ec 419 (imm) x Ec 423
(IMM) and Ec 421 (IMM) x Ec 420 (imm) we
raised nine and three zygotes, respectively, and these all showed a wild-type
germination pattern. A sporophyte from each cross (Ec 429 and Ec 430,
respectively) was raised and eight (for Ec 429) and 11 (for Ec 430) families
of gametophytes corresponding to single unilocular sporangia were isolated.
The mutant germination pattern was observed in the germlings from 
50% of
the 18-20 gametophytes analysed per family.
In none of the above analyses did a single gametophyte produce populations of partheno-sporophytes with both types of germination pattern.
The analyses of progeny from the three imm/IMM heterozygous
sporophytes (Ec 372, Ec 429 and Ec 430) were used to test for linkage between
IMM and the sex locus. For this, the phenotypes of the progeny from
each unilocular sporangium were used to determine the segregation pattern of
the two loci during the single meiotic event that had occurred in each
sporangium (see Table S2 in the supplementary material). No statistical
deviation from a 1:1:1:1 ratio was detected in any of the three crosses (e.g.
for strain Ec 372, 2=0.266, P>0.9), indicating
that IMM segregated independently of the sex locus.
Taken together, these data demonstrate that the imm mutation was stably inherited as a recessive Mendelian factor through several generations of the life cycle and that the IMM locus was not linked to the sex locus.
Gene expression in the sporophyte of the imm mutant indicates that it is partially converted into a gametophyte
The sporophyte generation of the imm mutant exhibited several
morphological features typical of the gametophyte generation during its early
development, such as asymmetric initial cell division, and yet remained
functionally a sporophyte (producing spores but not gametes). To determine
whether these similarities with the gametophyte generation were due to a
partial conversion of the sporophyte into a gametophyte, we assayed the
expression of two libraries of sporophyte- and gametophyte-upregulated genes
(isolated by suppression subtraction hybridisation) in the partheno-sporophyte
of the imm mutant using a microarray approach. cDNA fragment inserts
from 600 clones from each of the two SSH libraries were arrayed on glass
slides and hybridised with fluorescently labelled cDNA.
Fig. 7A shows that the relative
abundances of the transcripts corresponding to the sequences spotted on the
microarray were similar in two independent total RNA samples from wild-type
partheno-sporophytes of the strain Ec 432. By contrast, when cDNA from the
partheno-sporophyte stage of the imm mutant was compared with cDNA
from the same wild-type partheno-sporophyte, there was a marked tendency for a
subset of the transcripts corresponding to the gametophyte-expressed SSH
library to be more abundant in the mutant cDNA sample and for a subset of the
transcripts corresponding to the sporophyte-expressed SSH library to be less
abundant (Fig. 7B). A
statistical test, carried out using the Statistical Analysis of Microarrays
(SAM) method (Tusher et al.,
2001), identified 80 clones whose corresponding transcripts were
significantly more abundant in the imm partheno-sporophyte RNA sample
and 62 clones whose transcripts were significantly less abundant. Seventy-nine
of the 80 clones corresponding to genes upregulated in the imm mutant
(99%) were from the gametophyte-expressed SSH library, whereas 60 of the 62
clones corresponding to downregulated genes (97%) were from the
sporophyte-expressed SSH library (Fig.
7C). Taken together with the similarity between the early
morphogenesis of the imm mutant sporophyte and that of the wild-type
gametophyte (Fig. 3), these
data provide convincing evidence that there is a partial switch from the
sporophyte to the gametophyte developmental programme in the imm
mutant sporophyte.
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The results for the wild-type partheno-sporophyte and gametophyte samples showed that most of the IDW genes exhibited a sporophyte-specific or sporophyte-preferential pattern of expression, whereas most of the IUP genes exhibited a gametophyte-specific or gametophyte-preferential pattern of expression (Fig. 8). The difference between the expression levels in the two generations was very marked in most cases, with a maximum fold difference of 6975 for IUP9. Altogether, the quantitative PCR analysis allowed the validation of 16 genes (nine for the sporophyte and seven for the gametophyte) that showed a differential pattern of expression between the two generations (Fig. 8).
We compared the expression levels of these genes in the partheno-sporophyte and gametophyte of the imm mutant with the values obtained for the wild type (Fig. 8). With only a few exceptions (IDW7, IDW9, IDW11, IUP8 and IUP9), the genes were expressed at a similar level in imm and wild-type gametophytes. This is consistent with the fact that we did not detect any visible phenotype of the imm mutation in the gametophyte generation. In addition, in the imm partheno-sporophyte, most of the genes assayed showed an expression level that was either comparable with that of the wild-type gametophyte (IDW1, IDW2, IDW3, IUP1) or was intermediate between the abundances assayed in the two generations of the wild type (IDW4, IDW5, IDW6, IDW7, IDW8, IUP2, IUP3, IUP4, IUP5, IUP6, IUP7, IUP8, IUP9). For the last five genes, the transcripts were markedly less abundant in the imm partheno-sporophyte compared with the wild-type gametophyte but they were, nonetheless at least 28 times more abundant than in the wild-type partheno-sporophyte (see Table S4 in the supplementary material). The quantitative PCR, therefore, confirmed that there was an increase in the abundance of the transcripts of gametophyte-expressed genes and a decrease in the abundance of sporophyte-expressed genes in the imm sporophyte.
Quantitative PCR analyses were carried out on partheno-sporophytes because, as mentioned above, we observed no morphological or developmental differences between partheno-sporophytes and diploid sporophytes derived from gamete fusions. This was the case for both the wild-type Ec 32 strain and for the imm mutant. To further verify that the two stages of the life cycle behaved in the same manner, we also measured transcript abundances for five of the genes tested in Fig. 8 at both stages for both the wild type and the imm mutant. The results of this experiment (see Fig. S1 in the supplementary material) indicate, as expected, that partheno-sporophytes and zygote-derived sporophytes exhibit very similar patterns of gene expression and that the deregulation of generation-specific genes observed in the imm partheno-sporophyte also occurred in a imm/imm diploid sporophyte.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Explaining the stability of haploid-diploid life cycles has been
particularly problematic because, depending on the niche of the organism,
either the haploid or the diploid generation is expected to present certain
advantages and theoretical models predict that this should lead to a dominance
of one generation over the other under a wide range of conditions
(Mable and Otto, 1998). It has
been suggested, however, that if the two generations are adapted to different
ecological niches, this could stabilise a haploid-diploid life cycle (Stebbins
and Hill, 1980; Willson, 1981)
and this suggestion is supported by more recent theoretical work
(Hughes and Otto, 1999). It is
possible that the difference in morphology between the wild-type sporophyte
and gametophyte of Ectocarpus reflects an adaptation to different
ecological niches, with the dense, more robust thallus of the sporophyte,
particularly its prostrate base, being better adapted for persisting in less
favourable conditions during most of the year and the more fragile gametophyte
being short-lived and adapted for producing gametes over a period of a few
weeks. Future work on the ecology of the two generations of the
Ectocarpus life cycle might therefore provide a means to test
theoretical hypotheses concerning the stability of haploid-diploid life
cycles.
Phenotypic analysis of the imm mutant
The gametophyte generation of the imm mutant was phenotypically
wild type, but the sporophyte generation exhibited a gametophyte-like pattern
of germination and early development. Microarray analysis of gene expression
in the mutant strain showed that the morphological resemblance of the
imm sporophyte to a gametophyte was correlated with a specific
upregulation of a subset of the genes that are normally expressed during the
gametophyte generation and a corresponding downregulation of a subset of
sporophyte-generation genes. This analysis, therefore, provided evidence that
the mutant was affected in processes that are regulated during the life cycle.
In addition, phenotypic analysis of the imm mutant showed that the
development of mature sporophytes with the appropriate reproductive structures
(unilocular and plurilocular sporangia) was not contingent on the wild-type
sporophyte pattern of early development (involving symmetric division of the
initial cell and mediate differentiation). These two, temporally separate
parts of the sporophyte developmental programme were genetically uncoupled in
the imm mutant. Hence, not only does E. siliculosus exhibit
alternation between developmental programmes involving symmetric and
asymmetric initial cell divisions during the sporophyte and gametophyte phases
of the life cycle, respectively, but both symmetric (in the wild type) and
asymmetric (in imm individuals) initial cell divisions can give rise
to the sporophyte generation. Moreover, because the imm mutation
represents a single locus, germination and subsequent cell division patterns
might have been modified quite easily during evolution in the brown algal
lineage. This may help explain the variety of germination patterns observed in
different brown algal species.
Microarray analysis identified 40 genes whose level of expression was modified in the imm mutant. This number is an underestimate because only a small subset of the Ectocarpus gene set was represented on the array. These data suggest that IMM may be regulating a cascade of downstream genes to mediate its influence on early sporophyte development. Analysis of the sequences of the genes whose expression patterns were altered in the imm mutant provided only limited information about the molecular events underlying the observed phenotypes.
Current efforts aimed at developing E. siliculosus as a model
species (Peters et al., 2004a;
Coelho et al., 2007;
Charrier et al., 2008) are
expected to provide a means to investigate, at the molecular level, the
mechanisms behind the phenomena described in this study.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/8/1503/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Apt, K. E., Clendennen, S. K., Powers, D. A. and Grossman, A.
R. (1995). The gene family encoding the fucoxanthin
chlorophyll proteins from the brown alga Macrocystis pyrifera.
Mol. Gen. Genet. 246,455
-464.[CrossRef][Medline]
Berleth, T. and Chatfield, B. (2002).
Embryogenesis: pattern formation from a single cell. In The
Arabidopsis Book (ed. C. R. Somerville and E. M. Meyerowitz).
Rockville, MD: American Society of Plant Biologists.
Brownlee, C. and Bouget, F. Y. (1998). Polarity
determination in Fucus: from zygote to multicellular embryo.
Semin. Cell Dev. Biol.
9, 179-185.[CrossRef][Medline]
Charrier, B., Coelho, S. M., Le Bail, A., Tonon, T., Michel, G.,
Potin, P., Kloareg, B., Boyen, C., Peters, A. F. and Cock. J. M.
(2008). Development and physiology of the brown alga
Ectocarpus siliculosus: two centuries of research. Tansley Review.
New Phytol. 177,319
-332.[Medline]
Coelho, S. M., Peters, A. F., Charrier, B., Roze, D., Destombe,
C., Valero, M. and Cock, J. M. (2007). Complex life cycles of
multicellular eukaryotes: new approaches based on the use of model organisms.
Gene, 406,152
-170.[Medline]
Corellou, F., Coelho, S. M., Bouget, F. Y. and Brownlee, C.
(2005). Spatial re-organisation of cortical microtubules in vivo
during polarisation and asymmetric division of Fucus zygotes.
J. Cell Sci. 118,2723
-2734.
de Reviers, B. (2002). Biologie et
Phylogénie des Algues, tome 1. Paris: Belin.
de Reviers, B. (2003). Biologie et
Phylogénie des Algues, tome 2. Paris: Belin.
Fletcher, R. L. (1987). Seaweeds of
the British Isles Volume 3 Fucophyceae (Phaeophyceae), Part 1.
London, UK: British Museum (Natural History).
Fowler, J. E., Vejlupkova, Z., Goodner, B. W., Lu, G. and
Quatrano, R. S. (2004). Localization to the rhizoid tip
implicates a Fucus distichus Rho family GTPase in a conserved cell
polarity pathway. Planta
219,856
-866.[Medline]
Horvitz, H. R. and Herskowitz, I. (1992).
Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the
question. Cell 68,237
-255.[CrossRef][Medline]
Hughes, J. S. and Otto, S. P. (1999). Ecology
and the evolution of biphasic life cycles. Am. Nat.
154,306
-320.[CrossRef][Medline]
Huynh, J. R. and St Johnston, D. (2004). The
origin of asymmetry: early polarisation of the Drosophila germline
cyst and oocyte. Curr. Biol.
14,R438
-R449.[CrossRef][Medline]
Kawai, H., Motomura, T. and Okuda, K. (2005).
Isolation and purification techniques for macroalgae. In Algal
Culture Techniques (ed. R.A. Andersen), pp.133
-143. Amsterdam: Elsevier.
Kornmann, P. (1956). Über die Entwicklung
einer Ectocarpus confervoides-Form. Pubblicazioni della
stazione zoologica di Napoli 28,32
-43.
Mable, B. K. and Otto, S. P. (1998). The
evolution of life cycles with haploid and diploid phases.
BioEssays 20,453
-462.[CrossRef]
Morrison, S. J. and Kimble, J. (2006).
Asymmetric and symmetric stem-cell divisions in development and cancer.Nature
441,1068
-1074.[CrossRef][Medline]
Müller, D. G. (1964). Life-cycle of
Ectocarpus siliculosus from Naples, Italy.
Nature 203,1402
.
Müller, D. G. (1967). Generationswechsel,
Kernphasenwechsel und Sexualität der Braunalge Ectocarpus
siliculosus im Kulturversuch. Planta
75, 39-54.[CrossRef]
Müller, D. G. (1991). Mendelian
segregation of a virus genome during host meiosis in the marine brown alga
Ectocarpus siliculosus. J. Plant Physiol.
137,739
-743.
Pedersen, P. M. (1984). Studies on primitive
brown algae (Fucophyceae). Opera Botanica
74, 1-76.
Peters, A. F., Marie, D., Scornet, D., Kloareg, B. and Cock, J.
M. (2004a). Proposal of Ectocarpus siliculosus as a
model organism for brown algal genetics and genomics. J.
Phycol. 40,1079
-1088.[CrossRef]
Peters, A. F., Scornet, D., Müller, D. G., Kloareg, B. and
Cock, J. M. (2004b). Inheritance of organelles in artificial
hybrids of the isogamous multicellular chromist alga Ectocarpus
siliculosus (Phaeophyceae). Eur. J. Phycol.
39,235
-242.[CrossRef]
Quatrano, R. S. (1997). Cortical asymmetries
direct the establishment of cell polarity and the plane of cell division in
Fucus embryos. In Cold Spring Harbor Symposium on
Quantitative Biology, Vol. 62, pp.65
-70. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press.
Robinson, K. R. and Miller, B. J. (1997). The
coupling of cyclic GMP and photopolarization of Pelvetia zygotes.
Dev. Biol. 187,125
-130.[CrossRef][Medline]
Rychlik, W. and Rhoads, R. E. (1989). A
computer program for choosing optimal oligonucleotides for filter
hybridization, sequencing and in vitro amplification of DNA.
Nucleic Acids Res. 17,8543
-8551.
Scheres, B. and Benfey, P. N. (1999).
Asymmetric cell division in plants. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 50,505
-537.[CrossRef]
Schneider, S. Q. and Bowerman, B. (2003). Cell
polarity and the cytoskeleton in the Caenorhabditis elegans zygote.
Annu. Rev. Genet. 37,221
-249.[CrossRef][Medline]
Stache-Crain, B., Müller, D. G. and Goff, L. J.
(1997). Molecular systematics of Ectocarpus and
Kuckuckia (Ectocarpales, Phaeophyceae) inferred from phylogenetic
analysis of nuclear and plastid-encoded DNA sequences. J.
Phycol. 33,152
-168.[CrossRef]
Starr, R. C. and Zeikus, J. A. (1993). UTEX-The
culture collection of algae at the University of Texas at Austin.
J. Phycol. 29(Suppl), 1-106.[CrossRef]
Stebbins, G. L. and Hill, G. J. (1978). Did
multicellular plants invade the land? Am. Nat.
115,342
-353.[CrossRef]
Tusher, V. G., Tibshirani, R. and Chu, G.
(2001). Significance analysis of microarrays applied to the
ionizing radiation response. Proc. Natl. Acad. Sci.
USA 98,5116
-5121.
Vandesompele, J., De Prete, K., Pattyn, F., Poppe, B., Van Roy,
N., De Paepe, A. and Speleman, F. (2002). Accurate
normalization of real-time quantitative R.T-PCR data by geometric averaging of
multiple internal control genes. Gen. Biol.
3, 1-11.
Willson, M. F. (1981). On the evolution of
complex life cycles in plants: a review and an ecological perspective.
Ann. Missouri. Bot. Gard.
68,275
-300.[CrossRef]
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