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F.GENETIC
STRAINS RESEARCH CENTER
F-e. Plant Genetics Laboratory - Nori Kurata
Group
RESEARCH
ACTIVITIES
(1)
Molecular cytogenetics of plant meiosis analyzed
with rice mutants
Ken-ichi Nonomura, Mutsuko Nakano, Mitsugu
Eiguchi, Akio Miyao*, Hirohiko
Hirochika* and Nori Kurata
(*Natl. Inst. Agrobiol. Sci.)
--This study aims
to dissect genetic machinery controlling meiosis
and sporogenesis of higher plants. By screening of
insertional mutant lines tagged by the
Tos17, the endogenous retrotransposon of
rice, we could identify and isolate two meiotic
genes of interest; PAIR1 (HOMOLOGOUS
PAIRING ABERRATION IN
RICE MEIOSIS 1) (Nonomura et al. 2004a)
and PAIR2 (Nonomura et al. 2004b). The
PAIR2 gene encodes a protein of 610 amino
acids, homologous to the yeast HOP1 and
Arabidopsis ASY1. We raised a polyclonal
antibody against recombinant PAIR2 protein and
identified the PAIR2 expression in rice meiocytes
by western blotting and immuno-fluorescent
analyses. The PAIR2 protein began to accumulate in
the nucleus of meiocytes following the onset of
pre-meiotic DNA synthesis, and disappeared at
metaphase I. In early meiosis I, the PAIR2 antibody
stains the lateral elements, which are components
of chromosomal axes and play important roles in
mediating homologous chromosome pairing. The PAIR2
protein was removed from the chromosomes as soon as
the homologous chromatins aligned and paired. This
observation suggests the PAIR2 function in
recruiting the central components, which are
important to establish a rigid structure between
lateral elements of homologous partners.
--The
immuno-cytological analysis at diakinesis, prior to
metaphase I, revealed that the most PAIR2 proteins
were removed from chromosomes, but not from the
centromeric regions. The centromere-specific
histone H3 variant always colocalized with the
PAIR2 through early to late meiosis I. In addition,
our study revealed that the rice centromeres paired
preceding to usual homologous chromosome pairing.
We are now underway to make it sure that the
centromere pairs at early meiosis I prior to
pairing of other chromosomal regions in rice
meiocytes.
(2)
Structural and functional analysis of rice
OsHAP3 genes
Yukihiro Ito, Thiruvengadam Thirumurugan,
Kazumaru Miyoshi and Nori Kurata
--We identified 11
genes (OsHAP3A to OsHAP3K) which encode a
HAP3/NF-YB subunit of CCAAT-box binding complex in
rice by cDNA screening and a database search. We
showed that three genes, OsHAP3A,
OsHAP3B and OsHAP3C, are involved in
chloroplast biogenesis (Miyoshi et al. 2003). We
next examined the function of OsHAP3E, which
is most closely related to LEC1 and
L1L. The LEC1 and L1L are
Arabidopsis members of the HAP3/NF-YB
subunit and are critical regulators of
embryogenesis. The OsHAP3E was also
expressed in developing embryo and young panicle.
The antisense plants of OsHAP3E showed
slightly increased plant height, and its
overexpressing plants showed dwarfism, erected
leaves and abnormal inflorescence with the
increased number of spikelets. Microarray analysis
showed that brassinosteroid-related genes and
MADS-box genes were affected in the overexpressing
plants. These results suggested that OsHAP3E
may be involved in brassinosteroid-related response
and panicle development. Yeast two-hybrid analysis
showed that OsHAP3E interacted with several
specific members of OsHAP2 and OsHAP5, which are
other subunits of heterotrimeric CCAAT-box binding
complex. These results suggested that several
different combination of HAP complexes with OsHAP3E
can be formed in rice cells. Considering that
LEC1 and L1L are critical regulators
of embryogenesis in Arabidopsis,
OsHAP3A, B and C are
controlling factors of chloroplast biogenesis and
OsHAP3E is thought to regulate hormonal
and/or influorescence development pathways, plant
HAP3/NF-YB genes are suggested to have
diverse functions depending on each gene.
(3)
Regulation of expression of KNOX family
class 1 homeobox genes of rice
Yukihiro Ito and Nori Kurata
--KNOX
family class1 homeobox genes play a vital function
for shoot apical meristem (SAM) formation and
maintenance. We found that cytokinin, which is
necessary for shoot regeneration, induced the
expression of OSH1, a member of the
KNOX family, in the callus. Expression of
OSH1 was observed an hour after cytokinin
treatment, whereas expression of ORR6, a
homologue of a cytokinin early response gene, was
detected within 30 min. It was assumed that the
cytokinin-induced gene expression is under control
of a histidine phosphotransfer system which
consists of a histidine kinase, a
phosphotransmitter protein and a response
regulator. To examine this possibility, we
generated transgenic plants overexpressing
COS3 histidine kinase gene, OHP2
phosphotransmitter gene or ORR1 response
regulator gene. These transgenic plants showed
enhanced expression of OSH1 upon treatment
with cytokinin. This suggested that these genes are
involved in the cytokinin-induced OSH1
expression. Analysis of transgenic plants with
dominant negative constructs of these genes is
underway.
(4)
Generation and screening of enhancer trap lines of
rice
Yukihiro Ito and Nori Kurata
--To isolate
tissue-specific enhancers and valuable mutants
defective in various steps of rice development and
to clone the corresponding genes, we are generating
enhancer trap lines of rice. We employed an
enhancer trap system based on the Ac/Ds
two-element system and the GUS reporter gene. We
already have established the system by which
Ds-GUS transposants are efficiently
identified in rice (Ito et al. 2004).
--This year we
screened about 2,000 transposed lines and
identified 146 lines with tissue-specific GUS
activity. GUS expression was observed in various
organs including embryo (7 lines), leaf (59 lines),
root (22 lines), panicle (11 lines), flower (22
lines), seed (18 lines) and shoot apex (7 lines).
We also determined the Ds-GUS insertion site
of the 27 GUS-positive lines. We identified a
trapped gene by a Ds-GUS transposition in a
line P1772, which expressed the GUS on a leaf
lesion after wounding, as an example. In this line
the Ds-GUS was shown to be inserted near a
full-length cDNA AK073621 on chromosome 12, and
this gene was expressed transiently in the leaf
after wounding. This indicated that the enhancer of
this gene was trapped in P1772 and that our trap
system can usefully work not only on enhancer
selection but also on molecular identification of
the trapped genes. Cloning of other trapped
enhancers is underway. These data will be opened
through Oryzabase
(http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp).
(5)
Positional cloning of a segregation distortion gene
detected in a progeny of a cross between Japonica
and Indica rice
Yoshiaki Harushima and Nori Kurata
--The aim of this
study is isolation of the most prominent barrier on
chromosome 3 detected in the F2 of
Nipponbare-Kasalath hybrid by positional cloning,
and elucidation of the molecular nature of the
individual reproductive barrier. We have clarified
the pollen with Kasalath genotype at the
gametophyte gene preferentially fertilize eggs by
94% probability in the maternal plant that is
heterozygote or Kasalath homozygote at the
interactive locus on chromosome 6.
--For fine mapping the
gametophyte gene, we have selected plants with
recombination in the candidate region from 5691 F2
and 473 backcross plants and mapped it by their
selfed progeny test. A prominent causal gene is an
Argonaute gene. Expression of this
Argonaute gene in both Nipponbare pollen and
Kasalath pollen was confirmed. RT-PCR and
micro-array analysis showed there was no difference
in the expression level. However, high frequency of
nonsynonymous substitutions, 8 out of 9 single
nucleotide changes in a coding region, was
detected. This suggests rapid evolution that is
common feature of reproductive barriers. There are
two possibilities to explain Kasalath pollen
preferential fertilization; one is Kasalath pollen
gene accelerates fertilization, another is
Nipponbare pollen gene retards it in pollen
competition. For complementation test, two kinds of
Nipponbare near isogenic lines, both the aimed
gametophyte gene and the interactive gene regions
are Kasalath homozygote and only the interactive
gene region is Kasalath homozygote, were
transformed by the Nipponbare or Kasalath genomic
fragment containing the Argonaute with
GFP. These transformants are now growing and
their selfed seeds will be tested for the
transmission of introduced Argonaute and
GFP.
(6)
Identification of nuclear proteins showing various
sub-nuclear distribution in rice
Tadzunu Suzuki, Kazuki Moriguchi and Nori
Kurata
--We screend rice
nuclear proteins from three developmental stages;
young panicle, flowering panicle and regenerating
calli by using NTT (nuclear transportation trap)
system and identified over 500 genes including many
novel proteins and many transcription factors. A
subset of these genes were examined for their
nuclear localization by introducing GFP-fusion
constructs into onion epidermal cells. A variety of
protein localization in the cell; for instance,
nuclear peripheral, foci-like, speckle-like,
chromatin-associated and matrix-associated
distribution, were observed (Moriguchi et al.
2005). Some of these genes were revealed to be
expressed at specific stages of rice development by
RT-PCR and in situ hybridization. One of
these which we designated OsAHP1 was further
characterized for its tissue specificity and
sub-nuclear localization by detecting with an
antibody produced. The results showed that this
protein is expressed transiently in the floral
tissue and co-localized with chromatins in the
nucleus (Moriguchi et al. 2005).
(7)
Comparative genomics among cultivated and wild rice
species
Yukie Sano, Hiroyuki Kanamori*,
Nobukazu Namiki*, Yukiko Yamazaki and
Nori Kurata (*STAFF institute,
Tsukuba)
--We have started
to analyze gene structure and expression
differences between cultivated rice, Oryza
sativa (AA genome species), and wild rice of
O. punctata (BB genome) and O.
officinalis (CC genome). Total of 4,000 cDNA
clones randomly picked up from the libraries of SAM
(shoot apical meristem) or very early
influorescence were subjected to EST sequencing and
blast search analyses. Some clones were further
analyzed for full length clone sequences. Totally
about 2,900 clones have been qualified in their
sequences. Blast searches showed about 70% of the
clones had highly identical sequences with rice
cDNA or genomic sequences. Around 3% (from 7% to
0%) base substitution and in frame or out of frame
insertion/deletion were detected between cultivated
and wild rice in the clones which showed identities
with rice sequences. Simultaneous examination for
expression profiling using 22K microarrays revealed
scores of genes which showed quite large
differences in their expression between cultivated
and wild rice. We are going to identify gene
structure of clones having no similarity with rice
and showing much higher expression in either
cultivated or wild rice.
(8)
Rice genetic resource project in NBRP and
Oryzabase
Nori Kurata, Ken-ichi Nonomura, Yukihiro Ito and
Yukiko Yamazaki
--The National
Bioresource Project (NBRP) was organized in 2002 to
conserve and distribute biological resources for
scientific communities. Rice is one of the
organisms whose resources are required to amplify,
conserve and distribute to the scientific community
as genetic resources. In the NIG, we are dealing
2,000 wild rice accessions covering 10 genomes and
23 species collected all over the world for over 50
years. Four rice sub-centers in other institutions
dealing with different kinds of genetic materials
are joined to this project. Resource materials and
their information can be accessed at;
http://shigen.lab.nig.ac.jp/rice/oryzabase/nbrpStrains/aboutNbrp.jsp;jsessionid=
9AD3EACD512A1DA03ABB65874CF6381D.tomcat4_6
in the Oryzabase.
--The Oryzabase is a
comprehensive rice biological database composed of
15 sections of information. Major data
characteristic in the Oryzabase are; mutants and
their trait genes collection, wild rice collection
and development/anatomy of rice, together with
genetic maps, physical maps, basic biological data
and so on. The rice genetic resources committee
centered in the NIG is responsible to collect and
curate the contents of information and
construction, and maintenance of those data is
carried out by the genetic informatics laboratory.
The DB is accessible at;
http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp
Publications
Papers
1. Miyoshi, K., Ahn, B-O., Kawakatsu, T.,
Ito, Y., Itoh, J-I., Nagato, Y. and Kurata, N.
(2004). PLASTOCHRON1, a timekeeper of leaf
initiation in rice, encodes cytochrome P450.
Proc. Natl. Acad. Sci. USA 101, 875-880.
2. Ito, Y., Chujo, A., Eiguchi, M. and Kurata, N.
(2004). Radial axis differentiation in a globular
embryo is marked by HAZ, a PHD-finger
homeobox gene of rice. Gene 331, 9-15.
3. Nonomura, K.I., Nakano, M., Murata, K., Miyoshi,
K., Eiguchi, M., Miyao, A., Hirochika, H. and
Kurata, N. (2004). The insertional mutation of rice
PAIR2 gene, the ortholog of Arabidopsis
ASY1, caused a defect in homologous chromosome
pairing in meiosis. Mol. Genet. Genomics
271, 121-129.
4. Nonomura, K.I., Nakano, M., Fukuda, T., Eiguchi,
M., Miyao, A., Hirochika, H., Kurata, N. (2004).
The novel gene HOMOLOGOUS PAIRING ABERRATION IN
RICE MEIOSIS 1 of rice encodes a putative
coiled-coil protein required for homologous
chromosome pairing in meiosis. Plant Cell
16, 1008-1020.
5. Salina, E.A., Adonina, I., Vatolina, T. and
Kurata, N. (2004). A comparative analysis of the
composition and organization of two subtelomeric
repeat families of Aegilops speltoides
Tausch.and related species. Genetica 122,
227-237.
6. Moriguchi, K., Suzuki, T., Ito, Y., Yamazaki,
Y., Niwa, Y. and Kurata, N. (2005). Functional
isolation of novel nuclear proteins showing a
variety of sub-nuclear localizations. Plant Cell
17, 389-403.
Reviews
7. Itoh, J.I., Nonomura, K.I., Ikeda, K.,
Yamaki, S., Inukai, Y., Yamagishi, H., Kitano, H.
and Nagato, Y. (2005). Rice plant development: from
zygote to spikelet. Plant Cell Physiol. 46,
23-47.
8. Kurata, N., Miyoshi, K., Nonomura, K.I.,
Yamazaki, Y. and Ito, Y. (2005). Rice mutants and
genes related to organ development, morphogenesis
and physiological traits. Plant Cell Physiol.
46, 48-62.
Book
1.
野々村賢一・土井一行（2004）「生殖研究」,イネゲノム配列解読で何ができるのか（農業生物資源研+農文協,矢野昌裕・松岡信編）,90-102.
2.
倉田のり（2005）パキテン染色体標本,クロモソーム:植物染色体研究法（養賢堂,福井・向井・谷口編）印刷中.
ORAL
PRESENTATIONS
1. Kurata, N., Sano, Y., Eiguchi, M., Kanamori,
H., Yamazaki, Y. Comparative genomics of expressed
sequences between BB, CC and AA genomes in rice.
The 2nd international Symposium of rice functional
genomics. Tucson, Arizona USA, November 15-17,
2004
2.
伊藤幸博,永口貢,倉田のり「イネのエンハンサートラップ系統の作成とトラップされた遺伝子のクローニング」第106回日本育種学会,三重,2004年9月.
3.
野々村賢一・中野睦子・永口貢・宮尾安藝雄・広近洋彦・倉田のり「減数分裂期の染色体対合が欠損するイネpair1突然変異体の細胞学的解析」,日本育種学会第105回講演会、東京、2004年4月.
4.
野々村賢一「減数分裂染色体の相同性認識と生殖的隔離イネ分子遺伝学ワークショップ」,つくば、2004年7月.
5.
野々村賢一・中野睦子・永口貢・倉田のり「減数分裂の相同染色体対合を仲介するイネPAIR2タンパク質」,日本育種学会第106回講演会,三重,2004年9月.
6.
野々村賢一「相同染色体の対合を促進する減数分裂特異的なイネ遺伝子の解析」,文科省科研費・特定領域研究「植物自家不和合性」公開シンポジウム「植物の生殖研究―その最前線と今後の方向―」,東京大学,2004年11月.
POSTER
PRESENTATIONS
1.
伊藤幸博,三好一丸,倉田のり「イネPLA1遺伝子過剰発現体の解析」第45回日本植物生理学会,八王子,2004年3月.
2. Nonomura, K.I., Nakano, M., Miyao, A.,
Hirochika, H. and Kurata, N. (2004) Mutation
analyses for sporogenesis and meiosis using
retrotransposon-tagged lines of rice. Gordon
Conference, Plant Molecular Biology, Plymouth, NH,
USA, July.
3.
野々村賢一,中野睦子,永口貢,倉田のり「イネの減数分裂における相同染色体軸の構成タンパク質PAIR2の解析」第27回日本分子生物学会年会,神戸,2004年12月.
4.
伊藤幸博,倉田のり「サイトカイニンによるイネKNOXホメオボックス遺伝子の発現誘導」第27回日本分子生物学会年会,神戸,2004年12月.
EDUCATION
1. 倉田のり:北海道大学集中講義:札幌8月
2. 倉田のり:名古屋大学集中講義:名古屋11月
3. 倉田のり:東京大学集中講義:東京12月
SOCIAL
CONTRIBUTIONS
1. 倉田のり:日本学術会議育種学連携委員
2. 倉田のり:日本育種学会幹事
3. 倉田のり:日本育種学会学会賞選考委員
4. 倉田のり:生物遺伝資源イネ小委員会委員長
5. 倉田のり:農林水産省評価専門委員
6. 倉田のり:Rice Genetics Newsletter Editor
7. 倉田のり:NSF project advisory committee
member
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