DIVISION OF GENE EXPRESSION AND REGULATION I |
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Professor:
Research Associates:
Technical Staffs:
NIBB Research Fellow:
JSPS Postdoctoral Fellows:
Postdoctoral Fellows:
Visiting Scientist:
Graduate Students:)
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Technical Assistants:
Secretary: |
IIDA, Shigeru
TERADA, Rie
HOSHINO, Atsushi
TSUGANE, Kazuo
FURUKAWA, Kazuhiko
TANAKA-FUKADA, Sachiko
CHOI, Jeong-Doo
PARK, Kyeung-Il
EUN, Chan-Ho *
JOHZUKA-HISATOMI, Yasuyo
ISHIKAWA, Naoko **
KOUMURA, Toshiro ***
OHNISHI, Makoto 1)
IKEUE, Natsuko 2) ****
TAKAGI, Kyouko 3) *****
MORITA, Yasumasa
NAGAHARA, Miki
SAITOH, Miho
SANJO, Kazuko |
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* from October, 2003
** until March, 2003
*** from June, 2003
**** from April until November, 2003
***** from November, 2002
1) Graduate University for Advanced Studies
2) from Graduate School of Tokyo University
3) from Graduate School of Hokkaido University |
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The main interest of the group is in
understanding the biology of the dynamic genome, namely, genome organization
and reorganization and its impact on gene expression and regulation.
We are also characterizing various aspects of genetic and epigenetic
gene regulations particularly on flower pigmentation of morning glories.
In addition, we are undertaking reverse genetic approaches in order
to elucidate the nature of dynamic genome in plants.
I. Spontaneous mutants in morning glories.
Considerable attention has recently been paid to the morning glory
genus Ipomoea because of the experimental versatility of
its floral biology including the genetics of floral variation, flavonoid
biosynthesis, and transposon-induced mutations. The genus Ipomoea
includes about 600 species distributed on a worldwide scale that exhibit
various flower morphologies and pigmentation patterns. A large number
of Ipomoea species can be found in the Americas, particularly
in Mexico. Among the genus Ipomoea, three morning glories,
Ipomoea nil (the Japanese morning glory), Ipomoea purpurea
(the common morning glory), and Ipomoea tricolor, were domesticated
well as floricultural plants, and many mutants displaying various
flower pigmentation patterns were isolated. Among these morning glories,
I. nil and I. purpurea belong to the same subgenera
Ipomoea, whereas I. tricolor was classified into
another subgenera, Quamoclit. Both I. nil and I.
tricolor display blue flowers (see Figure 2A) that contain the
peonidin (3’ methoxyl cyanidin) derivative named Heavenly Blue
Anthocyanin (HBA), and I. purpurea produces dark purple flowers
containing a cyanidin derivative that lacks one glucose molecule and
a methyl residue from HBA. All of them produce dark-brown seeds. As
floricultural plants, spontaneous mutants of I. purpurea
and I. tricolor displaying either white or reddish flowers
were obtained while various spontaneous mutants of I. nil
exhibiting many different flower colorations were generated.
Although I. nil has long been believed to have originated
from Southeast Asia, a hypothesis that I. nil may have arrived
in Asia from tropical America was also recently proposed. In either
case, the plant had been introduced into Japan from China approximately
in the 8th Century as a medicinal herb. The seeds were used as a laxative,
and the plant became a traditional floricultural plant in Japan around
the 17th Century. I. nil has an extensive history of genetic
studies, and a number of its spontaneous mutants related to the color
and shape of the flowers have been isolated. It was also used extensively
for physiological studies of the photoperiodic induction of flowering.
The wild-type I. purpurea plant, which originated from Central
America, was probably introduced to Europe in the 17th Century, and
cultivars with white and red flowers were already recorded in the
late 18th Century. Like I. purpurea, I. tricolor originated from Central America, and several spontaneous mutants exhibiting
various flower pigmentations were obtained in the mid-20th Century.
As Figure 1 shows, the spontaneous mutants of I. nil, I.
purpurea, and I. tricolor carrying the magenta, pink, and fuchsia alleles, respectively, produce
reddish flowers containing pelargonidin derivatives, and all of them
are deficient in the gene for flavonoid 3’-hydroxylase (F3’H).
The magenta allele in I. nil is a nonsense mutation caused
by a single C to T base transition generating the stop codon TGA,
and the cultivar Violet carries the same mutation. Several tested pink mutants in I. purpurea carry inserts of the
0.55-kb DNA transposable element Tip201 belonging to the Ac/Ds superfamily at the identical site. No excision of Tip201 from the F3’H gene could be detected, and both splicing
and polyadenylation patterns of the F3’H transcripts
were affected by the Tip201 integration. The fuchsia allele in I. tricolor is a single T insertion generating
the stop codon TAG, and the accumulation of the F3’H transcripts was drastically reduced by the nonsense-mediated RNA decay.
The I. tricolor spontaneous mutant Blue Star carrying the
mutable ivory seed-variegated allele exhibits pale-blue flowers
with a few fine blue spots and ivory seeds with tiny dark-brown spots
(Figure 2). The mutable allele is caused by an intragenic tandem duplication
of 3.3 kb within a gene for transcriptional activator containing a
bHLH DNA-binding motif. Each of the tandem repeats is flanked by a
3-bp sequence AAT, indicating that the 3-bp microhomology is used
to generate the tandem duplication. The transcripts in the pale-blue
flower buds of the mutant contain an internal 583-bp tandem duplication
that results in the production of a truncated polypeptide lacking
the bHLH domain. The mRNA accumulation of most of the structural genes
encoding enzymes for anthocyanin biosynthesis in the flower buds of
the mutant was significantly reduced. The transcripts identical to
the wild-type mRNAs for the transcriptional activator were present
abundantly in blue spots of the variegated flowers, whereas the transcripts
containing the 583-bp tandem duplication were predominant in the pale-blue
background of the same flowers. The flower and seed variegations are
caused by somatic homologous recombination between an intragenic tandem
duplication in the bHLH gene, whereas various flower variegations
in Ipomoea are known to be caused by excision of DNA transposons inserted
into pigmentation genes.
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Fig.1. Flower phenotypes and structures of the
F3’H genes in I. nil, I. purpurea, and I.
tricolor exhibiting reddish flowers. A. I. nil
carrying the magenta allele. B. I.
purpurea carrying the pink allele. C.
I. tricolor carrying the fuchsia allele. |
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Fig.2. Flower and seed pigmentation phenotypes
and structures of the bHLH transcriptional regulatortor gene
for anthocyanin pigmentation in the I. tricolor wild-type
cultivar, Heavenly Blue (A and C),
and its mutant, Blue Star (B, D
and E). |
II. Targeted gene disruption by homologous recombination in rice.
Rice (Oryza sativa L.) is an important staple food for more
than half of the world’s population and a model plant for other
cereal species. We have developed a large-scale Agrobacterium-mediated
transformation procedure with a strong positive-negative selection
and succeeded in efficient and reproducible targeting of the Waxy
gene by homologous recombination without concomitant occurrence of
ectopic events, which must be an important first step for developing
a precise modification system of the genomic sequences in rice. By
improving our transformation procedure further, we are attempting
to modify Adh genes, which belong to a small multigene family
and reside adjacent to repetitive retroelements.
III. Characterization of mutable virescent
allele in rice.
Leaves of seedlings in the virescent mutant of rice are initially
pale yellow green due to partial deficient in chlorophyll and gradually
become green with the growth of the mutant. We have been characterizing
a spontaneous mutable virescent allele pale yellow leaf-variegated
(pyl-v), conferring pale yellow leaves with dark green sectors
in its seedlings (Figure 3). The pyl-v mutant was isolated
among progeny of a hybrid between indica and japonica
rice plants. The leaf variegation is regarded as a recurrent somatic
mutation from the recessive pale yellow allele to the dark green revertant
allele. The availability of the genomic sequences of both japonica
and indica subspecies facilitates map-based cloning of the
pyl-v allele. Mapping data indicate the mutation resides
in the short arm of the chromosome 3, and excision of a new DNA transposon
from the pyl gene appears to be responsible for conferring
the leaf variegation. It is important to emphasize here that tissue
culture is necessary in all of the currently available rice reverse
genetic approaches. No somaclonal variation is likely to occur in
mutant lines induced by our newly characterizing endogenous element,
because no tissue culture has been involved in its activation. Using
a newly identified DNA transposon, therefore, we are attempting to
develop a novel transposon tagging system in rice.
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Fig.3. A. Leaf phenotype of the mutable pyl-v
allele. B. Identification of the pyl-v
allele. The pyl-v allele was mapped between the
SSR markers RM251 and RM282 on chromosome 3 and subsequently
located between the SLP markers CH30524-1 and MMCAPS-1 on a
single BAC clone. The horizontal pentagonals represent predicted
genes. |
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Publication List:
Kikuchi, T., Nishimura, M., Hoshino, A., Morita, Y., Iida, S., Saito, N. and Honda, T. (2003) An efficient conversion of catechine into 3,4-trans-leucocyanidin. Heterocycles 60, 1469-1475.
Hoshino, A., Morita, Y., Choi, J.D., Saito, N., Toki, K., Tanaka,Y. and Iida, S. (2003) Spontaneous mutations of the flavonoid 3’-hydroxylase gene conferring reddish flowers in the three morning glories. Plant Cell Physiol. 44, 990-1001.
Yoshida, H., Akimoto, H., Yamaguchi, M., Shibata, M., Habu, Y., Iida, S. and Ozeki, Y. (2004) Alteration of methylation profiles in distinct cell lineages of the layers during vegetative propagation in carnation (Dianthus caryophyllus). Euphytica (in press).
Terada, R., Asao, H. and Iida, S. (2004) A Large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.). Plant Cell Reports (in press).
Iida, S., Morita, Y., Choi, J.D., Park, K.I. and Hoshino, A. (2004) Genetics and epigenetics in flower pigmentation associated with transposable elements in morning glories. Advances in Biophysics (in press). |
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