1 Department of Biological Sciences, Graduate School of Science, The University
of Tokyo, Tokyo 113-0033, Japan
2 National Institute for Basic Biology, Okazaki 444-8585, Japan
3 Department of Molecular Biomechanics, The Graduate University for Advanced
Studies, Okazaki 444-8585, Japan
* Author for correspondence (e-mail: mhasebe{at}nibb.ac.jp)
Accepted 19 January 2005
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SUMMARY |
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Key words: LEAFY, FLORICAULA, Fertilization, Zygote, Physcomitrella patens
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Introduction |
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The moss Physcomitrella patens has been recognized as a useful
model for the study of plant embryogenesis and development
(Cove et al., 1997;
Sakakibara et al., 2003
) for
several reasons. First, the exceptionally high efficiency of homologous
recombination in P. patens makes it the only land plant in which gene
targeting is feasible (Schaefer and
Zrÿd, 1997
). Second, the eggs, zygotes and young sporophytes
of P. patens are located solely in the cavity of its egg-bearing
organ, the archegonium, and are thus easily accessible for observation, while
those in seed plants are relatively difficult to observe. Third, P.
patens has a multicellular autotrophic body in its haploid generation,
permitting maintenance of mutants with severe defects in their diploid
generation.
While searching for genes involved in sporophyte development in P.
patens, we found that the homologs of the transcription factor
FLORICAULA/LEAFY (FLO/LFY) are required
for the first cell division in the zygote. FLO/LFY genes
have been reported only in land plants; a single copy or a few copies exist in
each genome (Himi et al.,
2001). In Arabidopsis thaliana, LFY plays a central role
in a gene network that controls flower development
(Ng and Yanofsky, 2000
).
LFY integrates environmental and endogenous signals that control the
timing of the transition from the vegetative to the reproductive phase
(Blázquez and Weigel,
2000
) and interacts with other transcription factors to activate
the transcription of floral homeotic genes that control the identity of floral
organs (Busch et al., 1999
;
Lamb et al., 2002
;
Lohmann et al., 2001
;
Parcy et al., 1998
;
Wagner et al., 1999
).
FLO/LFY functions are largely conserved in flowering plants,
even in distantly related species (Ahearn
et al., 2001
; Bomblies et al.,
2003
; Coen et al.,
1990
; Hofer et al.,
1997
; Molinero-Rosales et al.,
1999
; Schultz and Haughn,
1991
; Souer et al.,
1998
; Weigel et al.,
1992
). The functions of FLO/LFY genes are also
probably conserved in gymnosperms, including Pinus
(Mouradov et al., 1998
) and
Gnetum (Shindo et al.,
2001
). Although FLO/LFY homologs have been
cloned from several ferns and bryophytes
(Frohlich and Parker, 2000
;
Himi et al., 2001
), their
functions in these plants have not been characterized.
We report the characterization of two FLO/LFY genes in P. patens, and show that the FLO/LFY genes play a key role in the progression of the first cell division of the zygote. In addition, they are important for later sporophyte development.
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Materials and methods |
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Southern analyses
Genomic DNA was extracted from a mixture of protonemata and gametophores of
P. patens by the CTAB (Hexadecyltrimethyl-ammonium bromide) method
(Murray and Thompson, 1980).
The blotting and hybridization of 1 µg of digested genomic DNA were
performed as described previously (Shindo
et al., 2001
), using the gPpLFY1 and gPpLFY2 DNA fragments as
probes (Fig. 1A,B).
|
Construction of targeting plasmids and generation of transformants in P. patens
Based on the cDNA sequences of PpLFY1 and PpLFY2
(Himi et al., 2001), the
5' and 3' genomic regions of these cDNAs were amplified using
TAIL-PCR (Liu and Whittier,
1995
) and sequenced. These cDNA and genomic DNA sequences were
then used to construct targeting plasmids to make PpLFY1-GUS, PpLFY2-GUS,
PpFLY1-dis, PpLFY2-dis and PpLFY1-PpLFY2-dis lines in addition to
PpLFY ectopic expression lines in A. thaliana.
Genomic sequences of PpLFY1 and PpLFY2 were used to design and synthesize primer sets for the amplification of the majority of both PpLFY1 (2871 bp) and PpLFY2 (2852 bp) genomic DNA: L1-1int1 (5'-CTTGGCTTTAGACTCCTCTCATCGGTCG-3') and L1-3U1 (5'-TTGGTCAAGATCTCATGGCCGCAGCTG-3'), and PpLFY2-5' (5'-GCTCGGCTGCATTCCAGCTGCACTCTC-3') and PpLFY2-3' (5'-CTACAAAATAGTGTACAAGGGCTCATTCG-3'). Each amplified DNA fragment was cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), thereby generating pgPpLFY1 and pgPpLFY2.
A partial 1367 bp fragment of PpLFY1 genomic DNA ranging from the
second exon to just before the stop codon was also cloned into the pGEM-T
vector (Promega). A SalI-SmaI fragment was excised from this
plasmid and inserted into a SalI-ClaI site of the
pGUS-NPTII-1 plasmid that contained the coding sequence of uidA
(Jefferson et al., 1987), the
nopaline synthase polyadenylation signal (nos-ter), and an NPTII cassette
(Nishiyama et al., 2000
),
thereby creating an in-frame fusion of the PpLFY1 and uidA
genes. A 943 bp DNA fragment containing the 3' region of PpLFY1
gene, produced by TAIL-PCR using the gene-specific primer PpLFY-LA1
(5'-CGGATCTGGTATGTCCCCACGAAACTCC-3'), was inserted into the
SmaI site of this plasmid. The resulting plasmid was linearized with
BamHI and SalI for generation of the PpLFY1-GUS lines. Of 78
independent stable transformants, 18 were identified by PCR to have the fusion
construct in the PpLFY1 locus. Southern analyses of 12 of the 18
candidate lines showed that three lines (PpLFY1-GUS-1, 2 and 3) contained a
single insertion in the PpLFY1 locus (see Fig. S1A,C in the
supplementary material), while the other nine lines tested contained multiple
copies (data not shown).
A 1460 bp fragment containing a partial sequence of the 5' region of the PpLFY2 genomic DNA was amplified using the PpLFY2-5' and L2a-Hi3 (5'-CAAGCTTCGGCTTTCTCTGCCGGCCAGCGC-3') primers. A 1398 bp fragment of the 3' region of the PpLFY2 genomic DNA was amplified using the L2s-Ba1 (5'-CGGATCCGAAGGTTGAAGGAGCTATTCAAG-3') and PpLFY2-3' primers. These fragments were inserted into pGUS-NPTII-1, creating an in-frame fusion of the PpLFY2 and uidA genes. The resulting plasmid was linearized with NotI and SalI and used to generate the PpLFY2-GUS lines. Of 80 independent stable transformants, 44 were identified by PCR to have the fusion construct in the PpLFY2 locus. Southern analyses of 15 of the 44 candidate lines showed that 11 lines (PpLFY2-GUS-1 to -11) contained a single insertion in the PpLFY2 locus (see Fig. S1B,D in the supplementary material; data not shown), while the other lines tested contained multiple copies (data not shown).
To disrupt the PpLFY1 gene, the 5' SalI-HindIII fragment and the 3' XbaI fragment of pgPpLFY1 were inserted into the SalI-HindIII and XbaI sites of pHTS14, respectively. pHTS14 contains an HPT cassette composed of the CaMV 35S promoter, the hygromycin B phosphotransferase (hpt) gene, and the nos-ter sequence between multiple cloning sites. The resulting plasmid was used for transformation after linearization with NotI and then used to generate the PpLFY1-dis and PpLFY1-PpLFY2-dis lines. To generate PpLFY1-dis lines, 17 of 38 lines randomly selected from 81 independent stable transformants were identified by PCR as having a disrupted PpLFY1 locus (data not shown). Southern analyses of 10 of the 17 candidate lines showed that 8 lines (PpLFY1-dis-1 to 8) contained a single insertion in the PpLFY1 locus (see Fig. S2A,C in the supplementary material), while the other lines contained multiple copies (data not shown).
To disrupt the PpLFY2 gene, the fragment of PpLFY2 between the HindIII sites located in the second intron and the third exon of pgPpLFY2 was replaced with an NPTII cassette. The resulting plasmid was used for transformation after linearization with EcoRI and NotI. To generate the PpLFY2-dis line, 5 of 56 lines randomly selected from 79 independent stable transformants were identified by PCR as having a disrupted PpLFY2 locus (data not shown). Southern analyses of the 5 candidate lines showed that a single line (PpLFY2-dis-1) contained a single insertion in the PpLFY2 locus (see Fig. S2B,D in the supplementary material), while the other lines contained multiple copies (data not shown).
To generate double disruptants, an HPT cassette was inserted into the third exon of PpLFY1 in the PpLFY2-dis-1 line. Of 47 independent stable transformants, 20 were identified by PCR as having a disrupted PpLFY1 locus (data not shown). Southern analyses of 18 of the 20 candidate lines showed that 6 lines (PpLFY1-PpLFY2-dis-1 to -6) contained a single insertion in the PpLFY1 locus, in addition to the disrupted PpLFY2 locus (see Fig. S2D,E in the supplementary material), while the other lines contained multiple copies (data not shown).
Ectopic expression plasmids of PpLFY1 or PpLFY2 in A. thaliana
The PpLFY1 cDNA fragment was amplified by PCR using PpLFY-sense2
(5'-CCCCGGGGTTTGAATGGTGGACGACTGCCCTG-3') and PpLFY1-antisense1
(5'-ATGTACCCGGGCTCATTCACCGTGCTTC-3') primers. The PpLFY2
cDNA fragment was amplified with PpLFY-sense2 and PpLFY2-3'. Each DNA
fragment was cloned into the pBI121Hm-G vector, which was generated by
excising the uidA sequence from pBIH1-IG
(Akama et al., 1992) and which
contained the CaMV 35S promoter, the nos-ter, the hpt gene and the
nptII gene. These vectors were transferred into Agrobacterium
tumefaciens strain C58C1Rifr and used for transformation of
A. thaliana (Columbia).
Transformation of P. patens
Polyethylene glycol-mediated transformation was performed as described
previously (Nishiyama et al.,
2000). Stable transformants were further screened with PCR using
appropriate primer sets to confirm the insertion of the construct in each
locus. PCR-positive candidates were further analyzed by Southern analyses.
Confocal laser scanning microscopy
Shoot apices with gametangia and several young leaves were collected 3-4
weeks after the induction of gametangia and fixed in a solution of 4% (v/v)
glutaraldehyde and 1 µg/ml DAPI in 12.5 mM cacodylate (pH 6.9) overnight at
4°C (Christensen et al.,
1997). The fixed materials were then dehydrated in a graded
ethanol series and cleared in a 2:1 mixture of benzyl benzoate and benzyl
alcohol for observation on a Leica TCS SP2 confocal system equipped with a UV
laser (Leica, Wetzlar, Germany). Fluorescence between 400 and 700 nm was
detected under excitation with 351 nm and 364 nm excitation beams.
Sectioning and histochemical assay for GUS activity
For observation of the egg cells, the plant tissues were fixed in 5%
glutaraldehyde in phosphate buffer (pH 6.9) for 5 hours and in 2% osmium
tetroxide for 2 hours at room temperature, and then dehydrated with acetone
and embedded in Quetol 651 epoxy resin (Nisshin EM, Tokyo, Japan). Semi-thin,
0.5 µm sections were examined after staining with 0.5% (w/v) Toluidine Blue
O solution.
For observation of the sporophytes, the plant tissues were fixed in 4% (w/v) paraformaldehyde in cacodylate (pH 7.2) overnight at 4°C. After dehydration in an ethanol series, the samples were embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany). The 5 µm sections were examined after Toluidine Blue O staining.
The histochemical assay for GUS activity was performed as previously
described (Nishiyama et al.,
2000). The incubation time was adjusted from 2.5 to 12 hours,
depending on the tissues examined.
Genetic crosses
The wild-type and double disruptants were cultured on 30-mm diameter peat
pellets in separate plastic boxes. After cultivation at 25°C for 1 month,
followed by 3 weeks at 15°C, both antheridia and archegonia matured. A
peat pellet with wild-type plants and one with double disruptants were then
transferred into the same plastic box and the two peat pellets were submerged
in distilled water. After 30 seconds, the water above the mosses was decanted
and the culture was continued at 15°C for 5 weeks before harvesting
sporophytes.
The obtained spores were spread on BCDAT medium overlaid with a sheet of cellophane. After determining the germination rate of the spores, the cellophane was cut into quarters, and transferred to BCDAT medium containing either no antibiotics or hygromycin (30 mg/l), G418 (20 mg/l), or both hygromycin (30 mg/l) and G418 (20 mg/l).
Ectopic expression of PpLFY1 or PpLFY2 in A. thaliana
Transformations of A. thaliana (Columbia) using
35S::PpLFY1 and 35S::PpLFY2 (see above) were performed by
the floral dip method (Clough and Bent,
1998). The T3 seedlings of transformed A. thaliana were
grown at 22°C under conditions of 16 hours light/8 hours dark or 10 hours
light/14 hours dark. The 35S::LFY7-7 and 35S::LFY16-7 lines
are subsequent generations of 35S::LFY7 and 35S::LFY16,
respectively (Shindo et al.,
2001
).
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Results |
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PpLFY1 and PpLFY2 show similar expression patterns
Analysis by semi-quantitative reverse transcription-polymerase chain
reaction (RT-PCR) revealed higher expression levels of PpLFY1 and
PpLFY2 in gametophores and sporophytes than in protonemata
(Fig. 2). To investigate
spatiotemporal expression patterns in more detail, the uidA gene,
which codes for ß-glucuronidase (GUS), was inserted just before the stop
codon of PpLFY1 (Fig.
1D), or in-frame in the second exon of PpLFY2
(Fig. 1E). Multiple single
insertion lines were identified for each construct (see Fig. S1 in the
supplementary material).
|
|
Double disruption of PpLFY genes causes defects in sporophyte formation
To investigate the functions of PpLFY1 and PpLFY2,
disruptants for each gene and for both genes were generated
(Fig. 1F,G; see Fig. S2 in the
supplementary material). For PpLFY1, 8 lines (PpLFY1-dis-1 to 8)
contained a single insertion of an HPT cassette, which confers resistance to
hygromycin, in the PpLFY1 locus. For PpLFY2, one line
(PpLFY2-dis-1) contained a single insertion of an NPTII cassette, which
confers G418 resistance, in the PpLFY2 locus. In addition to the
PpLFY2-dis-1 line, PpLFY2 was disrupted in the PpLFY2-GUS lines
(Fig. 1E), whose phenotypes
were also observed.
PpLFY1 PpLFY2 double disruptants were generated by disrupting PpLFY1 in the background of the PpLFY2-dis-1 line. Six lines (PpLFY1-PpLFY2-dis-1 to 6) with a single insertion in the PpLFY1 locus, in addition to the disrupted PpLFY2 locus were isolated.
The morphology of the gametophytes in all single and double disruptants was not distinguishable from that of the wild type. However, the percentage of gametophores that developed sporophytes was significantly decreased in these disruptant lines (Table 1, Fig. 4). The decrease was very slight in the PpLFY2-dis and PpLFY2-GUS lines, but severe in the PpLFY1-dis and PpLFY1-PpLFY2-dis lines. Two phenotypic classes were recognized among the PpLFY1-dis lines; PpLFY1-dis-2, 6 and 7 showed an intermediate decrease in sporophyte development and PpLFY1-dis-1, 3 and 8 showed a severe decrease. The two types of phenotypes caused by the same gene disruption are unusual and further studies are necessary. The sporophytes in the double disruptants had abnormal morphology, which is described below.
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Crosses between the wild type and the double disruptants
To examine whether the PpLFY1-PpLFY2-dis lines can form normal,
fertilizable egg cells, the PpLFY1-PpLFY2-dis lines, as the female parents,
were crossed with the wild type, as the male parent. Among 140 sporophytes
obtained, 135 sporophytes had abnormal morphology and/or the germination rates
of the spores were close to zero. The rare protonemata that germinated were
resistant to both hygromycin and G418, indicating that these sporophytes were
not formed by a cross between the double disruptant and the wild type. Five of
the 140 sporophytes obtained had morphology that was indistinguishable from
that of the wild type, and the germination rates of these spores were largely
similar to that of the wild type (Table
3). Approximately half of the protonemata that germinated from
these spores were resistant to either hygromycin or G418, and approximately a
quarter was resistant to both drugs. These frequencies were consistent with
the expected values if these five sporophytes resulted from a cross between
double disruptant eggs and wild-type sperm.
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|
Constitutive expression of PpLFY1 and PpLFY2 in A. thaliana
In contrast to the moss, there is not a zygotic phenotype in
flo/lfy mutants of angiosperm
(Bomblies et al., 2003;
Coen et al., 1990
;
Hofer et al., 1997
;
Molinero-Rosales et al., 1999
;
Shultz and Haughn, 1991; Souer et al.,
1998
; Weigel et al.,
1992
). To learn whether the function of FLO/LFY genes in
flowering plants has diverged from that of PpLFY genes, we generated
A. thaliana plants that constitutively expressed PpLFY1 or
PpLFY2. Constitutive expression of A. thaliana LFY mRNA
under the control of the CaMV 35S promoter has been shown to induce early
flowering and the formation of terminal flowers
(Weigel and Nilsson, 1995
;
Blázquez et al., 1997
;
Blázquez and Weigel,
2000
). Expression of PpLFY mRNAs in A. thaliana
plants transformed with 35S::PpLFY1 and 35S::PpLFY2
constructs was verified by northern analyses (data not shown). We could not
examine whether transcribed mRNAs were translated because of the lack of PpLFY
antibodies. Further studies are necessary to confirm PpLFYs are translated in
A. thaliana, although unusual codons in A. thaliana were not
found in PpLFYs (data not shown). Under either long- or short-day
conditions, the 35S::PpLFY plants flowered at the same time as the
wild type and there was no abnormal phenotype (see Table S1 in the
supplementary material). Control experiments, in which the A. thaliana
LFY genes were constitutively expressed, showed early flowering and
terminal flowers.
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Discussion |
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A change in fluorescence appears to indicate fertilization
(Fig. 5), similar to what has
been reported in some flowering plants
(Hoshino et al., 2004;
Webb and Gunning, 1994
;
Zhou, 1987
). By this
criterion, fertilization was seemingly normal in the PpLFY
disruptants. After fertilization, however, the putative zygote of the double
disruptants remained in the single-cell stage, indicating that the PpLFY
proteins are important for the first cell division of the zygote, along with
the normal development of the zygote, including the cellular expansion and the
accompanying extensive vacuolation. Since LFY protein from A.
thaliana has been shown to be a DNA-binding transcription factor
(Parcy et al., 1998
), the
PpLFY gene products may regulate the expression of genes responsible
for the zygote development and the zygotic cell division.
Possible involvement of PpLFY genes in sporophyte development
The few sporophytes formed by the PpLFY1-PpLFY2-dis double disruptants and
the severe PpLFY-1 single disruptants had abnormal morphology. These
sporophytes were possibly generated by parthenogenesis, which is common in the
three species of Physcomitrium under laboratory culture conditions
(Lal, 1984). The mode of
emergence of sporophytes from the archegonium venter and the multiple
sporangia occasionally observed in the disruptant lines are reminiscent of the
parthenogenetic sporophytes of Physcomitrium cyathicarpum
(Fig. 6E,F). Abnormal
sporophytes were not observed in the wild-type plants, most likely because the
sporophytes resulting from fertilization develop much earlier than those
resulting from parthenogenesis. Normally, a single sporophyte matures on the
apex of a gametophore, even though several archegonia are fertilized,
suggesting that the early growth of a sporophyte inhibits the development of
the embryos on the same gametophore apex that were fertilized later. In the
PpLFY disruptant lines, normal sporophytes resulting from
fertilization do not grow, and thus sporophytes arising from parthenogenesis
can survive and grow.
In addition to having the morphological characteristics commonly observed
in sporophytes that arise from parthenogenesis, the PpLFY disruptant
sporophytes had other abnormalities. The sporangia of the disruptants were
much softer than those of the wild type and often withered during development
(Fig. 6G). The number of
differentiated spores present in each sporangium varied in the disruptants.
Germinations occurred in only a few spores of several tens to several
thousands of spores per sporangium. This is in contrast to the case in
parthenogenetic sporophytes, which regularly form viable spores
(Lal, 1984). These additional
abnormal phenotypes may be related to the loss of PpLFY gene function
and may indicate that the PpLFY genes are involved in sporophyte
development in addition to zygote development. The expression of both
PpLFY mRNAs and proteins in the developing sporophytes (Figs
2,
3) is concordant with this
hypothesis.
The sporophytes of the PpLFY double disruptants exhibited
abnormalities in the patterns of cell division, but apparently not in
organogenesis. Normal sporophytes consist of three organs: the sporangium, the
seta and the foot (Fig. 3X);
all of these organs were formed even in the severely defective sporophytes
(Fig. 6G,I-K). An aberrant
pattern of cell division is the probable cause of the abnormalities in cell
number, cell shape and cell arrangement in the sporophytes of disruptants
(Fig. 6H-K). These
abnormalities are reminiscent of those observed in fass mutants of
A. thaliana (Torres-Ruiz and
Jürgens, 1994), which showed defects in the pattern of cell
division but not in organogenesis during embryogenesis. This may suggest that
the PpLFY proteins regulate the transcription of genes involved in cell
division in the sporophytes. Whether the decreased spore germination rates in
the double disruptants are caused by a defect in mitosis of sporogenous cells
or by a defect in meiosis of the spore mother cells, or both, will be the
subject of future research. Our results also suggest that cell division is
regulated differently in sporophytes and gametophytes in P. patens,
because the protonemata and gametophores of the PpLFY disruptants had
no morphological differences from the wild type.
Evolution of FLO/LFY genes and body plan in land plants
The developmental processes from gametogenesis to the first cell division
of the zygote commonly occur in both unicellular and multicellular organisms
with sexual reproduction, and also probably occurred in the unicellular common
ancestor of land plants and metazoans. This suggests that at least some of the
molecular mechanisms involved in the developmental processes should be shared
among all organisms. However, FLO/LFY genes have been found
only in land plants and the involvement of the PpLFY genes in the
initiation of the first cell division in the zygote suggests that plants have
evolved molecular mechanisms that differ from those of animals, even for very
early stages of development. This is concordant with the fact that plant cells
have a unique mode of cytokinesis involving cell plate formation.
The FLO/LFY genes of flowering plants, such as the A.
thaliana LFY gene, function in the transition from the vegetative to the
reproductive phase (Blázquez et al.,
1997; Weigel and Nilsson,
1995
), where one of its principal targets is APETALA1
(AP1) (Liljegren et al.,
1999
; Parcy et al.,
1998
; Wagner et al.,
1999
). In addition, LFY activates floral homeotic genes
and their cofactors (Busch et al., 2001;
Lamb et al., 2002
;
Parcy et al., 1998
;
Schmid et al., 2003
;
Weigel and Meyerowitz, 1993
;
William et al., 2004
). These
functions are generally preserved in other angiosperms, although
FLO/LFY has acquired additional roles in some plants such as pea
(Ahearn et al., 2001
;
Bomblies et al., 2003
;
Coen et al., 1990
;
Hofer et al., 1997
;
Molinero-Rosales et al., 1999
;
Schultz and Haughn, 1991
;
Souer et al., 1998
;
Weigel et al., 1992
).
Gymnosperm LFY homologs from Gnetum (GpLFY) and
Pinus (NEEDLY) are predominantly expressed in reproductive
meristems (Mouradov et al.,
1998
; Shindo et al.,
2001
), and the overexpression of GpLFY or NEEDLY
complements LFY function in the lfy null mutant and enhances
early flowering, similar to that observed with overexpression of LFY.
Therefore, these gymnosperm homologs probably have similar functions to those
of the A. thaliana LFY gene. These findings suggest that the common
ancestor of gymnosperms and angiosperms had a FLO/LFY gene
with similar functions to those of the A. thaliana LFY gene. The fern
LFY homologs CrLFY are also predominantly expressed in the
reproductive meristems, and not in the vegetative ones, but their expression
patterns are more restricted than those of the five fern MADS-box genes
(Hasebe et al., 1998
;
Himi et al., 2001
). This may
imply that the CrLFY genes have a function in the transition from
vegetative to reproductive growth, but that they are not indispensable for the
induction of the fern MADS-box genes analyzed.
In contrast to the partial or full functional conservation of the
FLO/LFY genes among vascular plants, the
FLO/LFY homologs in the moss P. patens function
differently from those of vascular plants. Although the PpLFY genes
function in the sporophytic generation, as is the case in vascular plants,
LFY and its homologs that have been reported in vascular plants do
not function in embryogenesis. Ectopic overexpression of PpLFY genes
did not cause early flowering in A. thaliana (see Table S1 in the
supplementary material), and PpLFY fused with A. thaliana
LFY promoter did not complement the lfy mutant
(Maizel et al., 2005). These
observations suggest that the function of the FLO/LFY genes
in the transition from the vegetative to the reproductive phase and in the
induction of the floral homeotic MADS-box genes evolved in the vascular plant
lineage after the divergence of mosses.
The basic body plans and the functions of signaling molecules in metazoans
are well conserved among a wide variety of lineages from Hydra to
vertebrates (Carroll et al.,
2001; Hobmayer et al.,
2000
). Green plants emerged on land approximately 480 million
years ago, and the moss lineage diverged from the vascular plant lineage
almost immediately after that time, which is more recent than the Cambrian
explosion, when most of the major lineages of extant metazoans diverged. The
FLO/LFY homologs are key developmental regulators in both
vascular plants and mosses; the differences in the functions of these genes in
the two lineages suggest that the body plans of land plants diverged
unexpectedly.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/7/1727/DC1
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahearn, K. P., Johnson, H. A., Weigel, D. and Wagner, D. R.
(2001). NFL1, a Nicotiana tabacum LEAFY-like
gene, controls meristem initiation and floral structure. Plant Cell
Physiol. 42,1130
-1139.
Akama, K., Shiraishi, H., Ohta, S., Nakamura, K., Okada, K. and Shimura, Y. (1992). Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains. Plant Cell Rep. 12,7 -11.
Ashton, N. W. and Cove, D. J. (1977). The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss Physcomitrella patens. Mol. Gen. Genet. 154,87 -95.[CrossRef]
Blázquez, M. A. and Weigel, D. (2000). Integration of floral inductive signals in Arabidopsis.Nature 404,889 -892.[CrossRef][Medline]
Blázquez, M. A., Soowal, L. N., Lee, I. and Weigel,
D. (1997). LEAFY expression and flower initiation in
Arabidopsis. Development
124,3835
-3844.
Bomblies, K., Wang, R.-L., Ambrose, B. A., Schmidt, R. J.,
Meeley, R. B. and Doebley, J. (2003). Duplicate
FLORICAULA/LEAFY homologs zfl1 and zfl2
control inflorescence architecture and flower patterning in maize.
Development 130,2385
-2395.
Busch, M. A., Bomblies, K. and Weigel, D.
(1999). Activation of a floral homeotic gene in Arabidopsis.Science 285,585
-587.
Carroll, S. B., Grenier, J. K. and Weatherbee, S. D. (2001). From DNA to Diversity. Massachusetts: Blackwell Science, Inc.
Christensen, C. A., King, E. J., Jordan, J. R. and Drews, G. N. (1997). Megagametogenesis in Arabidopsis wild type and the Gf mutant. Sex. Plant Reprod. 10, 49-64.[CrossRef]
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735 -743.[CrossRef][Medline]
Coen, E. S., Romero, J. M., Doyle, S., Elliott, R., Murphy, G. and Carpeter, R. (1990). floricaula: A homeotic gene required for flower development in Antirrhinum majus. Cell 63,1311 -1322.[CrossRef][Medline]
Cove, D. J., Knight, C. D. and Lamparter, T. (1997). Mosses as model systems. Trends Plant Sci. 2,99 -105.[CrossRef]
Frohlich, M. W. and Meyerowitz, E. M. (1997). The search for flower homeotic gene homologs in basal angiosperms and Gnetales: a potential new source of data on the evolutionary origin of flowers. Int. J. Plant Sci. 158,S131 -S142.[CrossRef]
Frohlich, M. W. and Parker, D. S. (2000). The mostly male theory of flower evolutionary origins: from genes to fossils. Syst. Bot. 25,155 -170.
Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W., Muller, P., Delbarre, A., Ueda, T., Nakano, A. and Jürgens, G. (2003). The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112,219 -230.[CrossRef][Medline]
Hasebe, M., Wen, C.-K., Kato, M. and Banks, J. A.
(1998). Characterization of MADS homeotic genes in the fern
Ceratopteris richardii. Proc. Natl. Acad. Sci. USA
95,6222
-6227.
Himi, S., Sano, R., Nishiyama, T., Tanahashi, T., Kato, M., Ueda, K. and Hasebe, M. (2001). Evolution of MADS-box gene induction by FLO/LFY genes. J. Mol. Evol. 53,387 -393.[CrossRef][Medline]
Hobmayer, B., Rentzsch, F., Kuhn, K., Happel, C. M., von Laue, C. C., Snyder, P., Rothbächer, U. and Holstein, T. W. (2000). WNT signalling molecules act in axis formation in the diploblastic metazoan Hydra. Nature 407,186 -189.[CrossRef][Medline]
Hofer, J., Turner, L., Hellens, R., Ambrose, M., Matthews, P., Michael, A. and Ellis, N. (1997). UNIFOLIATA regulates leaf and flower morphogenesis in pea. Curr. Biol. 7,581 -587.[CrossRef][Medline]
Hoshino, Y., Scholten, S., von Wiegen, P., Lörz, H. and Kranz, E. (2004). Fertilization-induced changes in the microtubular architecture of the maize egg cell and zygote an immunocytochemical approach adapted to single cells. Sex. Plant Reprod. 17,89 -95.
Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W. (1987). GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6,3901 -3907.[Abstract]
Lal, M. (1984). The culture of bryophytes including apogamy, apospory, parthenogenesis and protoplasts. In The Experimental Biology of Bryophytes (ed. A. F. Dyer and J. G. Duckett), pp. 97-115. London: Academic Press.
Lamb, R. S., Hill, T. A., Tan, Q. K.-G. and Irish, V. F.
(2002). Regulation of APETALA3 floral homeotic gene
expression by meristem identity genes. Development
129,2079
-2086.
Liljegren, S. J., Gustafson-Brown, C., Pinyopich, A., Ditta, G.
S. and Yanofsky, M. F. (1999). Interactions among
APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate.
Plant Cell 11,1007
-1018.
Liu, Y.-G. and Whittier, R. F. (1995). Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25,674 -681.[CrossRef][Medline]
Lohmann, J. U., Hong, R. L., Hobe, M., Busch, M. A., Parcy, F., Simon, R. and Weigel, D. (2001). A molecular link between stem cell regulation and floral patterning in Arabidopsis.Cell 105,793 -803.[CrossRef][Medline]
Maizel, A., Busch, M. A., Tanahashi, T., Perkovic, J., Kato, M., Hasebe, M. and Weigel, D. (2005). Molecular evolution of the floral regulator LEAFY by substitutions in the DNA binding domain. Science (in press).
Mayer, U., Büttner, G. and Jürgens, G.
(1993). Apical-basal pattern formation in the
Arabidopsis embryo: studies on the role of the gnom gene.
Development 117,149
-162.
Molinero-Rosales, N., Jamilena, M., Zurita, S., Gómez, P., Capel, J. and Lozano, R. (1999). FALSIFLORA, the tomato orthologue of FLORICAULA and LEAFY, controls flowering time and floral meristem identity. Plant J. 20,685 -693.[CrossRef][Medline]
Mouradov, A., Glassick, T., Hamdorf, B., Murphy, L., Fowler, B.,
Marla, S. and Teasdale, R. D. (1998). NEEDLY, a
Pinus radiata ortholog of FLORICAULA/LEAFY genes, expressed
in both reproductive and vegetative meristems. Proc. Natl. Acad.
Sci. USA 95,6537
-6542.
Murray, M. G. and Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8,4321 -4325.[Abstract]
Ng, M. and Yanofsky, M. F. (2000). Three ways to learn the ABCs. Curr. Opin. Plant Biol. 3, 47-52.[CrossRef][Medline]
Nishiyama, T., Hiwatashi, Y., Sakakibara, K., Kato, M. and Hasebe, M. (2000). Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res. 7,9 -17.[Medline]
Parcy, F., Nilsson, O., Busch, M. A., Lee, I. and Weigel, D. (1998). A genetic framework for floral patterning. Nature 395,561 -566.[CrossRef][Medline]
Reski, R. (1998). Development, genetics and molecular biology of mosses. Bot. Acta 111, 1-15.
Sakakibara, K., Nishiyama, T., Sumikawa, N., Kofuji, R., Murata,
T. and Hasebe, M. (2003). Involvement of auxin and a
homeodomain-leucine zipper I gene in rhizoid development of the moss
Physcomitrella patens. Development
130,4835
-4846.
Schaefer, D. G. and Zrÿd, J.-P. (1997). Efficient gene targeting in the moss Physcomitrella patens. Plant J. 11,1195 -1206.[CrossRef][Medline]
Schmid, M., Uhlenhaut, N. H., Godard, F., Demar, M., Bressan,
R., Weigel, D. and Lohmann, J. U. (2003). Dissection
of floral induction pathways using global expression analysis.
Development 130,6001
-6012.
Schultz, E. A. and Haughn, G. W. (1991).
LEAFY, a homeotic gene that regulates inflorescence development in
Arabidopsis. Plant Cell
3, 771-781.
Shindo, S., Sakakibara, K., Sano, R., Ueda, K. and Hasebe, M. (2001). Characterization of a FLORICAULA/LEAFY homologue of Gnetum parvifolium and its implications for the evolution of reproductive organs in seed plants. Int. J. Plant Sci. 162,1199 -1209.[CrossRef]
Souer, E., van der Krol, A., Kloos, D., Spelt, C., Bliek, M.,
Mol, J. and Koes, R. (1998). Genetic control of
branching and floral identity during Petunia inflorescence
development. Development
125,733
-742.
Torres-Ruiz, R. A. and Jürgens, G. (1994).
Mutations in the FASS gene uncouple pattern formation and
morphogenesis in Arabidopsis development.
Development 120,2967
-2978.
Wagner, D., Sablowski, R. W. M. and Meyerowitz, E. M.
(1999). Transcriptional activation of APETALA1 by LEAFY.
Science 285,582
-584.
Wardlaw, C. W. (1955). Embryogenesis in Plants. London: Methuen & Co. Ltd.
Webb, M. C. and Gunning, B. E. S. (1994). Cell biology of embryo sac development in Arabidopsis. In Genetic Control of Self-Incompatibility and Reproductive Development in Flowering Plants (ed. Williams, E. G. Clarke, A. E. and Knox, R. B.), pp. 461-485. Dordrecht: Kluwer academic publishers.
Weigel, D. and Meyerowitz, E. M. (1993). Activation of floral homeotic genes in Arabidopsis.Science 261,1723 -1726.
Weigel, D. and Nilsson, O. (1995). A developmental switch sufficient for flower initiation in diverse plants. Nature 377,495 -500.[CrossRef][Medline]
Weigel, D., Alvarez, J., Smyth, D. R., Yanofsky, M. F. and Meyerowitz, E. M. (1992). LEAFY controls floral meristem identity in Arabidopsis. Cell 69,843 -859.[CrossRef][Medline]
William, D. A., Su, Y. H., Smith, M. R., Lu, M., Baldwin, D. A.
and Wagner, D. (2004). Genomic identification of
direct target genes of LEAFY. Proc. Natl. Acad. Sci.
USA 101,1775
-1780.
Zhou, C. (1987). A study of fertilization events in living embryo sacs isolated from sunflower ovules. Plant Sci. 52,147 -151.[CrossRef]