DIVISION OF SPECIATION MECHANISMS I |
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Professor:
Associate Professor:
Research Associates:
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Secretary: |
HASEBE,
Mitsuyasu
MURATA, Takashi
FUJITA, Tomomichi
HIWATASHI, Yuji (Oct. 1~)
SUMIKAWA, Naomi
HIWATASHI, Yuji (~Sept. 30)
NISHIYAMA, Tomoaki (Sept. 16~)
SATO, Yoshikatsu
NISHIYAMA, Tomoaki (~Sept. 15)
AONO, Naoki (April 1~)
MIYAZAKI, Saori (May 19~)
KOBAYASHI-ARAKAWA, Satoko (~Sept. 30)
SAKAKIBARA, Keiko(~March 31)
NAKAMURA, Tohru (Niigata Univ.) (~March 31)
KITANI, Masakazu (April 1~)
TANIKAWA, Yukiko
BITOH, Yoshimi
NARUSE, Mayumi
AOKI, Etsuko
OONO, Chikako (Feb. 1~)
SUZUKI, Yoriko (Feb. 1~)
Paul G. Wolf 1) (Sept. 22~Dec. 22)
Carol A. Rowe 1) (Sept. 22~Dec. 22)
KABEYA, Kazuko |
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1) from Utah State University, USA
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All living organisms evolved from a
common ancestor that lived more than 3.5 billion years ago, and the
accumulation of mutations in their genomes has resulted in the present
biodiversity. Traces of the evolutionary process are found in the
genomes of extant organisms. By comparing the gene sequences and gene
networks of different organisms, we can infer (1) the phylogenetic
relationships of extant organisms and (2) the genetic changes that
caused the evolution of morphology and development. The inferred phylogenetic
relationships provide important insights into problems in various
fields of evolutionary biology. Our group focuses on biogeography,
the evolution of morphological traits, and systematics in a wide range
of taxa. Concerning the evolution of morphology and development, we
hope to explore the genetic changes that led to the evolution of the
plant body plan. We have selected Arabidopsis (angiosperm), Gnetum
(gymnosperm), Ginkgo (gymnosperm), Ceratopteris (pteridophyte), Physcomitrella
(bryophyte), and some green algae as models to compare the functions
of genes involved in the development of both reproductive and vegetative
organs in land plants.
I. Origin of the Plant Cell
The first green alga cell evolved via symbiosis between an ancestral
non-photosynthetic eukaryote and a cyanobacterium. Cyanobacteria now
exist as chloroplasts in the host cell. The factors and mechanisms
of chloroplast movement are being investigated to reveal the molecular
mechanisms used to "domesticate" cyanobacteria as organelles.
Analyses of cytosolic calcium icon concentration and cytoskeleton
organization during chloroplast movement in the moss Physcomitrella
patens is in progress by a team directed by Y. Sato.
II. Evolution from unicellular to multicellular
organisms
The first evolutionary step from unicellular to multicellular organisms
is to form two different cells from a single cell via asymmetric cell
division. The first cell division of a protoplast isolated from the
protonemata of the moss Physcomitrella patens is asymmetric
regarding to its shape and nature, and gives rise to an apical meristematic
cell and a differentiated non-meristematic cell. A systematic overexpression
screening for genes involved in asymmetric cell division of protoplasts
in P. patens is in progress by a team directed by T. Fujita.
We constructed three full-length cDNA libraries from non-treated,
auxin-treated, and cytokinin-treated protonemal cells of P. patens,
then determined the sequences of more than 40,000 cDNAs from the both
ends (Nishiyama, Fujita et al. 2003). We used these clones as materials
for the overexpression screening. Individual cDNAs were selected based
on their sequence, subcloned under a constitutive promoter and introduced
into the protoplasts of P. patens for transient expression.
We observed and categorized phenotypes of the regenerating protoplasts.
Thus far we identified many cDNAs, whose overexpression resulted in
symmetric cell division rather than asymmetric cell division, isotropic
outgrowth with no polarity or curved cells showing incorrect direction
of growth. These preliminary results indicate that some of these genes
likely function for polarity formation and/or asymmetric cell division
of the protoplasts. Functional analyses of these genes should help
us to understand molecular mechanisms of how plants generate distinct
cell lineages to build their multicellular bodies.
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(Figure 1) Asymmetric cell division of a Physcomitrella
protoplast. Regular asymmetric cell division (1, 2) and defective
cell division with overexpression (3, 4). Cell wall of the cell
in (1) is visualized with calcoflor in (2). |
III. Evolution from cells to tissues
The most prominent difference between plant and animal cells is that
plant cells have a cell wall and do not move during development. Therefore,
the plane of cell division and the direction of cell elongation, which
are regulated by cortical microtubules, determine the morphology of
differentiated tissues and organs.
Organization of g-tubulin
g-tubulin is a protein that is essential
for the formation of microtubules in animal cells. We found that g-tubulin
is located at the end of cortical microtubules, and a loss of g-tubulin
due to gene silencing causes a malformed organ with irregularly shaped
cells. In vitro experiments using isolated plasma membrane/microtubule
complexes suggested that g-tubulin attaches
onto the side of existing cortical microtubules, and initiates a new
cortical microtubule from it. The nucleus is known to initiate microtubules
after cell division. We hypothesize that microtubules formed around
the nucleus elongate to cell surface, and trigger initiation of cortical
microtubules via attachment of g-tubulin.
Once cortical microtubules are formed, they can turnover without microtubules
from the nucleus. Factor(s) responsible for attachment of g-tubulin
onto the side of microtubules is a key element responsible for the
difference between plant and animal cells. Isolation of the factor(s)
responsible for attachment of g-tubulin
onto the side of microtubules by biochemical and other approaches
is inprogress by a team directed by T. Murata.
Dynamics of actin filaments and microtubules in the moss P.
patens
Cells of the moss P. patens gametophytes are an excellent
model to study dynamics of cytoskeleton because of the easiness for
observation and the feasibility of gene-targeting. Transformants with
reporter constructs by fusing a GFP with P. patens a-tubulin
or an actin binding domain of mouse talin were established by Y. Sato
and collaborators to visualize microtubules and actin filaments. These
transformants will be useful to investigate the dynamics of microtubules
and actin filaments in the processes of cell division, cell elongation,
and chloroplast movement.
IV. Evolution of molecular mechanisms in the development
of vegetative organs
Meristem initiation and maintenance
Postembryonic growth of land plants occurs from the meristem, a localized
region that gives rise to all adult structures. Meristems control
the continuous development of plant organs by balancing the maintenance
and proliferation of stem cells, and directing their differentiation.
Meristem initiation and maintenance is a fundamental question in plant
development research. However, the molecular mechanisms involved in
meristem initiation and maintenance have not been studied in detail
because most loss-of-function mutants are lethal. In the moss Physcomitrella
patens, the developmental process of meristem is well defined
at the cellular level, and gene targeting based on homologous recombination
is feasible. Thus, meristem development in P. patens is used
as a model system for studies of meristem development in land plants.
We established approximately 20,000 gene- or enhancer-trap lines in
P. patens to clone genes involved in meristem development.
Seven lines, exhibiting reporter gene (uidA) expression preferentially
in the apical cells, were isolated. Corresponding genes in three of
these trap lines were identified as encoding kinesin- and ubiquitin-like
proteins, and an unknown protein. Disruption of the gene encoding
ubiquitin-like protein suggests that the gene be involved in cell
division and elongation through microtubule organization. The functions
of other genes in the meristem are currently under investigation by
a team directed by Y. Hiwatashi.
The morphology of the shoot meristem in land plants varies. To investigate
whether the molecular mechanisms of shoot development in angiosperms
are conserved in other land plants, the functions of the KNOX homeobox,
and the ZWILLE, NAC, and PIN genes, which are indispensable for shoot
meristem development in angiosperms, are being studied in the fern
Ceratopteris and the moss Physcomitrella.
Rhizoid differentiation
Rhizoids are multicellular filaments with similar functions to root
hairs, and are observed in a wide range of plants. A team directed
by K. Sakakibara, who was a graduate student in our lab, examined
mechanisms underlying rhizoid development in the moss, Physcomitrella
patens (Sakakinara et al. 2003).
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Figure. 2 (1) a gametophyte of P. patens.
(2) an auxin-treated gametophyte with plenty of rhizoids. |
V. Origin and evolution of floral homeotic genes
The flower is the reproductive organ in angiosperms, and floral homeotic
genes, such as MADS-box genes and FLO/LEAFY genes, regulate floral
organ identity. To investigate the origin of floral homeotic genes,
the functions of MADS-box genes and the FLO/LEAFY genes in gymnosperms
(Gnetum, Ginkgo, and cycads), a fern (Ceratopteris),
a moss (Physcomitrella), and three green algae (Chara,
Coleochaete, and Closterium) are being analyzed.
Land plants are believed to have evolved from a gametophyte (haploid)-dominant
ancestor without a multicellular sporophyte (diploid plant body);
most genes expressed in the sporophyte probably originated from those
used in the gametophyte during the evolution of land plants. To analyze
the evolution and diversification of MADS-box genes in land plants,
gametophytic MADS-box genes were screened using macroarray analyses
for 105 MADS-box genes found in the Arabidopsis genome (Kofuji et
al. 2003). Eight MADS-box genes were predominantly expressed in pollen,
male gametophyte, and analyses of their function are in progress by
a team directed by N. Aono.
VI. Evolution of life cycles
The mosses and flowering plants diverged more than 400 million years
ago. The mosses have haploid-dominant life cycles, while the flowering
plants are diploid-dominant. The common ancestors of land plants are
inferred to have been haploid-dominant, suggesting that genes used
in the diploid body of flowering plants were recruited from the genes
used in the haploid body of their ancestors during the evolution of
land plants.
To assess the evolutionary hypothesis that genes used in diploid body
of flowering plants were recruited from the genes used in haploid
body of the ancestors, a team directed by T. Fujita constructed an
expressed sequence tag (EST) library of Physcomitrella, and T. Nishiyama
mainly worked on the comparison of the moss transcriptome to the genome
of Arabidopsis. We constructed full-length enriched cDNA libraries
from auxin-treated, cytokinin-treated, and untreated gametophytes
of Physcomitrella, and sequenced both ends of more than 40,000 clones.
These data, together with the mRNA sequences in the public databases,
were assembled into 15,883 putative transcripts. Sequence comparisons
of Arabidopsis and Physcomitrella showed that the haploid transcriptome
of Physcomitrella appears to be quite similar to the Arabidopsis genome,
supporting the evolutionary hypothesis. Our study also revealed that
a number of genes are moss specific and were lost in flowering plant
lineage. Our full-length cDNA library will be a good resource for
functional genomic studies in plants. Our EST data together with some
information on the moss is open to public at PHYSCObase (http://moss.nibb.ac.jp).
VII. Molecular mechanisms of speciation
Reproductive isolation is the first step in speciation. To obtain
insight into reproductive isolation, several receptors specifically
expressed in the pollen tube are being studied to screen for the receptors
that are involved in pollen tube guidance by a team directed by S.
Miyazaki.
Polyploidization is a major mode of speciation in plants, although
the changes that occur after genome duplication are not well known.
Polyploid species are usually larger than diploids, but the mechanisms
responsible for the size difference are unknown. To investigate these
mechanisms, tetraploid Arabidopsis was established and its gene expression
patterns are being compared to those of diploid wild-type plants using
microarrays.
VIII. Phylogenetic analysis of land plants
We determined the complete nucleotide sequence of the chloroplast
genome of the leptosporangiate fern, Adiantum capillus-veneris L. (Pteridaceae) as a collaboration work with P. Wolf and C. Rowe
from Utah State University (Wolf et al. 2003). Phylogenetic analysis
of basal land plants using the sequence is in progress by T. Nishiyama.
Many unusual start codons and internal stop codons were found, suggesting
that extensive number of RNA editing exists in the genome.
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Publication List:
Kofuji, R., Sumikawa, N., Yamasaki, M., Kondo, K., Ueda, K., Ito, M. and Hasebe, M. 2003. Evolution and divergence of MADS-box gene family based on genome wide expression analyses. Mol. Biol. Evol. 20: 1963-1977.
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.
Nishiyama, T., Fujita, T., Shin-I, T., Seki, M., Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P., Hayashizaki, Y., Shinozaki, K., Kohara, Y., and Hasebe, M. 2003. Comparative genomics of Physcomitrella patens gemetophytic transcriptome and Arabidopsis thaliana: Implication for land plant evolution. Proc. Natl. Acad. Sci. USA 100: 8007-8012.
Wolf, P.G., Rowe, C.A., Sinclair, R.B., and Hasebe, M. 2003. Complete nucleotide sequence of the chloroplast genome from a leptosporangiate fern, Adiantum capillus-veneris L. DNA Res. 10: 59-65.
Tanabe, Y., Uchida, M. Hasebe, M., and Ito, M. 2003. Characterization of the Selaginella remotifolia MADS-box gene. J. Plant Res. 116: 71-75.
Itoh, Y., Hasebe, M., Davies, E., Takeda, J., and Ozeki, Y. 2003. Survival of Tdc transposable elements of the En/Spm superfamily in the carrot genome. Mol. Gen. Genomics 269: 49-59.
Rivadavia, F., Kondo, K., Kato, M., and Hasebe, M. 2003. Phylogeny of the sundews, Drosera (Droseraceae) based on chloroplast rbcL and nuclear 18S ribosomal DNA sequences. Amer. J. Bot. 90: 123-130. |
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