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Junshi Yazaki, Zenpei Shimatani, Akiko Hashimoto, Yuko Nagata, Fumiko Fujii,

Keiichi Kojima, Kohji Suzuki, Toshiki Taya, Mio Tonouchi, Charles Nelson, Allen
Nakagawa, Yasuhiro Otomo, Kazuo Murakami, Kenichi Matsubara, Jun Kawai,
Piero Carninci, Yoshihide Hayashizaki and Shoshi Kikuchi
Physiol Genomics 17:87-100, 2004. First published Feb 24, 2004;
doi:10.1152/physiolgenomics.00201.2003

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Whole plant responses, key processes, and adaptation to drought stress: the case of rice
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J. Exp. Bot., January 1, 2007; 58 (2): 169-175.
[Abstract] [Full Text] [PDF]

Gibberellin Regulates Mitochondrial Pyruvate Dehydrogenase Activity in Rice


A. Jan, H. Nakamura, H. Handa, H. Ichikawa, H. Matsumoto and S. Komatsu
Plant Cell Physiol., February 1, 2006; 47 (2): 244-253.
[Abstract] [Full Text] [PDF]

Global Patterns of Gene Expression in the Aleurone of Wild-Type and dwarf1 Mutant Rice

P. C. Bethke, Y.-s. Hwang, T. Zhu and R. L. Jones


Plant Physiology, February 1, 2006; 140 (2): 484-498.
[Abstract] [Full Text] [PDF]

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Physiological Genomics publishes results of a wide variety of studies from human and from informative model systems with
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July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by
the American Physiological Society. ISSN: 1094-8341, ESSN: 1531-2267. Visit our website at http://www.the-aps.org/.
Physiol Genomics 17: 87–100, 2004.
First published February 24, 2004; 10.1152/physiolgenomics.00201.2003.

CALL FOR PAPERS Comparative Genomics

Transcriptional profiling of genes responsive to abscisic acid and gibberellin


in rice: phenotyping and comparative analysis between rice and Arabidopsis
Junshi Yazaki,1 Zenpei Shimatani,2 Akiko Hashimoto,2 Yuko Nagata,2 Fumiko Fujii,1
Keiichi Kojima,3 Kohji Suzuki,3 Toshiki Taya,4 Mio Tonouchi,4 Charles Nelson,5
Allen Nakagawa,5 Yasuhiro Otomo,6 Kazuo Murakami,6 Kenichi Matsubara,6,7
Jun Kawai,8 Piero Carninci,9 Yoshihide Hayashizaki,8,9 and Shoshi Kikuchi1
1
Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;
2
Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan;
3
Hitachi Software Engineering, Tokyo 140-0002, Japan; 4Agilent Technologies, Tokyo 190-0033, Japan; 5Agilent
Technologies, Palo Alto, California 94304; 6Laboratory of Genome Sequencing and Analysis Group, Foundation for the

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Advancement of International Science, Tsukuba, Ibaraki 305-0062, Japan; 7Nara Institute of Science and Technology, Ikoma,
Nara 630-0101, Japan; 8Laboratory for Genome Exploration Research Group, Institute of Physical and Chemical Research
(RIKEN) Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Kanagawa 230-0045, Japan;
and 9Genome Science Laboratory, RIKEN, Hirosawa, Wako, Saitama 351-0198, Japan
Submitted 3 December 2003; accepted in final form 6 February 2004

Yazaki, Junshi, Zenpei Shimatani, Akiko Hashimoto, Yuko genome-wide expression analysis; germination; dormancy; compara-
Nagata, Fumiko Fujii, Keiichi Kojima, Kohji Suzuki, Toshiki tive genomics; cis-element
Taya, Mio Tonouchi, Charles Nelson, Allen Nakagawa, Yasuhiro
Otomo, Kazuo Murakami, Kenichi Matsubara, Jun Kawai, Piero
Carninci, Yoshihide Hayashizaki, and Shoshi Kikuchi. Transcrip- RICE (Oryza sativa) is an important food crop that is useful as
tional profiling of genes responsive to abscisic acid and gibberellin in an experimental model because of its small genome size,
rice: phenotyping and comparative analysis between rice and Arabi- extensive genetic map, relative ease of transformation, and
dopsis. Physiol Genomics 17: 87–100, 2004. First published February synteny with other cereal crops. Draft sequences of the O.
24, 2004; 10.1152/physiolgenomics.00201.2003.—We collected and sativa L. ssp. indica (41) and japonica (8) genomes, obtained
completely sequenced 32,127 full-length complementary DNA clones
from Oryza sativa L. ssp. japonica cv. “Nipponbare.” Mapping of
by the whole-genome shotgun sequencing method, have been
these clones to genomic DNA revealed ⬃20,500 transcriptional units published. The National Institute of Agrobiological Sciences
(TUs) in the rice genome. For each TU, we selected 60-mers using an (NIAS) at Tsukuba, Japan, and its collaborators have con-
algorithm that took into account some DNA conditions such as base structed useful tools for functional genomics through the Rice
composition and sequence complexity. Using in situ synthesis tech- Genome Project of Japan. These tools consist of 32,127 full-
nology, we constructed oligonucleotide arrays with these TUs on glass length cDNA (FL-cDNA) clones (18), over 600 expression
slides. We targeted RNAs prepared from normally grown rice callus profiles developed by using an 8,987-cDNA microarray (39), a
and from callus treated with abscisic acid (ABA) or gibberellin (GA). high-quality genomic sequence that has 99.99% accuracy (5,
We identified 200 ABA-responsive and 301 GA-responsive genes, 32), a genetic map with 3,267 DNA markers (9), rice material
many of which had never before been annotated as ABA or GA for genetic analysis, and about 50,000 transposon insertion
responsive in other expression analysis. Comparison of these genes
revealed antagonistic regulation of almost all by both hormones; these
lines (24). All the information from these tools can be accessed
had previously been annotated as being responsible for protein storage through the Rice PIPELINE system (40). These tools may be
and defense against pathogens. Comparison of the cis-elements of important for improving not only rice but also other cereals,
genes responsive to one or antagonistic to both hormones revealed because functionally important sequences are conserved and
that the antagonistic genes had cis-elements related to ABA and GA may be identified by their synteny. Of these tools, the FL-
responses. The genes responsive to only one hormone were rich in cDNA clones, in particular, are necessary for the identification
cis-elements that supported ABA and GA responses. In a search for of exon-intron boundaries and gene-coding regions within
the phenotypes of mutants in which a retrotransposon was inserted in genomic sequences and for comprehensive gene function anal-
these hormone-responsive genes, we identified phenotypes related to ysis at transcriptional and translational levels. On the basis of
seed formation or plant height, including sterility, vivipary, and the results of our large-scale FL-cDNA analysis (18), we have
dwarfism. In comparison of cis-elements for hormone response genes
between rice and Arabidopsis thaliana, we identified cis-elements for
constructed a monitoring system that uses an oligonucleotide
dehydration-stress response as Arabidopsis specific and for protein array to monitor gene transcriptional levels and to develop
storage as rice specific. genome-wide functional analysis of rice. The array was com-
posed of 21,938 probes with 60-mer oligonucleotides synthe-
sized at a gene-specific region (2, 13, 34) from 32,127 FL-
Article published online before print. See web site for date of publication cDNAs. Mapping of these cDNA clones to genomic DNA
(http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: S. Kikuchi, National
revealed that there are about 20,500 transcriptional units (TUs),
Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki and clustering of these cDNA clones revealed a unique clone
305-8602, Japan (E-mail: skikuchi@nias.affrc.go.jp). set. Of the ⬃20,500 TUs located on genomic DNA, single
1094-8341/04 $5.00 Copyright © 2004 the American Physiological Society 87
88 PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE

clones on TU are ⬃14,000 and multiple clones on TU are Plant Material and RNA Preparation
⬃16,000 (⬃6,500 TUs). About 2,000 clones were unmapped The callus used for total RNA extraction was derived from the
according to incomplete genome sequences. The probes were scutellum of the japonica rice cultivar “Nipponbare” and was culti-
selected from these results. vated in Murashige and Skoog medium (27) containing 10 ␮M
We used the arrays as probes to hybridize target RNAs 2,4-dichlorophenoxyacetic acid. Such callus maintains the ability to
prepared from normally grown callus and from callus treated develop roots and leaves. After the calluses had been cultured in the
with abscisic acid (ABA) or gibberellin (GA). The interaction medium for 30 days, they were transferred to a medium containing the
between ABA and GA is an important factor controlling the plant hormones ABA or GA and cultured for 3 days. The concentra-
transition from embryogenesis to germination in seeds. The tion of each plant hormone was adjusted to 50 ␮M. After culturing,
effects of these hormones are competitive, in that ABA pro- we used an RNeasy Plant Mini Kit (Qiagen, Tokyo, Japan) to extract
total RNA from the hormone-treated calluses and from the control
motes seed dormancy, whereas GA promotes seed germina-
calluses (which were not treated with either hormone for the 3-day
tion. Cereals are excellent plants in which to explore the period). mRNA was isolated with an Oligotex-dt30 (Super) mRNA
molecular mechanisms involved in hormonally regulated gene purification kit (Takara, Shiga, Japan). Purified mRNA was amplified,
expression, particularly the antagonism between ABA and GA labeled, and hybridized to the rice 22,000-oligonucleotide array ac-
(14). Reports of such explorations (10, 22) have suggested the cording to the manufacturer’s protocols (Agilent Technologies). For
presence of cross talk, but these studies have investigated only each experiment described in this study, the data presented represent

Downloaded from physiolgenomics.physiology.org on November 28, 2008


part of the relationship between dormancy and germination in averaged results from hybridization to four oligonucleotide arrays in
plants. two-dye swap experiments.
In a previous study we elucidated the mechanisms of inter- Data Scanning, Quantification, and Processing
action between ABA and GA signaling in rice by using the
above tools for rice functional genomics, including rice 8,987- The hybridized and washed material on each glass slide was
cDNA microarray (38). Here we describe a comprehensive scanned with an Agilent DNA microarray scanner (model G2565BA;
transcriptional profiling of phytohormone response genes in Agilent Technologies). Feature Extraction and Image Analysis soft-
rice, phenotype analysis, and comparative analysis of transcrip- ware (version A.6.1.1, Agilent Technologies), was used to locate and
delineate every spot in the array and to integrate each spot’s intensity,
tional regulators between rice and Arabidopsis, for which we filtering, and normalization by using the LOWESS (also know as
used a more comprehensive and specific 21 938-oligonucleo- “LOESS”) method (36, 37). From the four replications of each
tide array. experiment we calculated the average ratio of expression of each spot
after dividing the signal intensity of mRNA from hormone-treated
MATERIALS AND METHODS callus by the signal intensity of that from untreated callus; these data
were then subjected to LOWESS normalization. All of the expression
Rice FL-cDNA Clones profiles are available as gene series 661 (GSE661), including gene
sample 9853 (GSM9853), GSM9854, GSM9855, GSM9856,
All 32,127 FL-cDNA clones were generated by large-scale FL- GSM9857, GSM9858, GSM9859, and GSM9860 on gene platform
cDNA analysis as part of the Rice Full-length cDNA Project, which 477 (GPL477) in the Gene Expression Omnibus (GEO) at the Na-
is a collaborative effort of NIAS, Foundation for the Advancement of tional Center for Biotechnology Information (NCBI; http://
International Science, and Institute of Physical and Chemical Re- www.ncbi.nlm.nih.gov/geo/).
search (RIKEN) under the supervision of the Bio-oriented Technol-
ogy Research Advancement Institution (BRAIN) (18). A homology Cis-Element Search in Upstream Region of Rice Genes
search was performed with BLASTN and BLASTX, and by homology
searches of publicly available sequence data we assigned tentative After we had mapped the FL-cDNA links with the 22,000-oligo-
protein functions. We used 21,938 TUs that were equivalent to nucleotide array on the rice genome, we performed a cis-element
⬃32,000 FL-cDNA clones; to maximize the effective use of these search. From the 5⬘-end sequences of the FL-cDNAs, the promoter
⬃32,000 published clones, in accordance with the recommendations sequences were obtained by comparison with the rice genomic se-
of the manufacturer of oligonucleotide array (Agilent Technologies, quences. We selected 1,000 bp of genomic sequences upstream from
Tokyo, Japan), the array format was 22,000 spots per glass slide. The the 5⬘ terminus of each FL-cDNA clone by using the data from the
functional annotations and identities (including accession numbers TIGR Rice Genome Project (http://www.tigr.org/tdb/e2k1/osa1/
and related reference papers) of all ⬃32,000 rice FL-cDNA clones are BACmapping/description.shtml) and searched for about total 300
listed in the Knowledge-based Oryza Molecular Biological Encyclo- cis-elements known in plants by using the PLACE cis-element data-
pedia (KOME; http://cdna01.dna.affrc.go.jp/cDNA/). base (11) (http://www.dna.affrc.go.jp/htdocs/PLACE/).
Specification of Cis-Elements for Hormone-Responsive Genes
Rice High-Density Oligonucleotide Array Construction in Rice
The array was composed of 21,938 probes with 60-mer oligonu- The cis-elements for hormone-responsive genes were compared
cleotides synthesized at a gene-specific region from 28,469 FL- among the gene groups in comparisons 1 and 2 below, and the types
cDNAs. Mapping of these cDNA clones to genomic DNA revealed of cis-element of each gene group were characterized.
that there are ⬃20,500 TU, and clustering of these cDNA clones Comparison 1. We compared cis-elements among genes that were
revealed a unique clone set. The probes were selected by means of upregulated by ABA, downregulated by GA, and both upregulated by
these results. For each of the clones, we selected the top 60-mers by ABA and downregulated by GA. For each of these three groups of
using an algorithm that took into account binding energy, base genes, we divided the total number of each type of cis-element in each
composition, sequence complexity, cross-hybridization binding en- group by the number of genes in each group. We then compared the
ergy, and secondary structure (2, 13, 34). We constructed the oligo- numbers of each type of cis-element per gene among the three groups.
nucleotide arrays with these TUs on glass slides by using in situ Comparison 2. We compared cis-elements among genes that were
synthesis technology under a customized contract with Agilent Tech- upregulated by GA, downregulated by ABA, and both upregulated by
nologies (2, 13, 34). GA and downregulated by ABA. By the same method used in

Physiol Genomics • VOL 17 • www.physiolgenomics.org


PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE 89
comparison 1, we compared the numbers of cis-elements per gene expression were contained in the area of the median (0.00)
among the three groups. We selected cis-elements that were present in ⫾3SD over a normal distribution. Of the 200 genes selected,
at least two genes, and the cis-elements of each group were charac- 110 were upregulated and 90 were downregulated by ABA;
terized.
altered expression levels were identified from the expression
Cis-Element Search for Genes of Arabidopsis thaliana and profile of the analysis of the four oligonucleotide array analysis
Specification of Cis-Element for Rice and A. thaliana in two-dye swap experiments. Sixty-two of the 200 genes had
functional annotations in a BLASTN search of the National
The identifiers (IDs) of the Munich Information Center for Protein Center for Biotechnology Information (NCBI; ⬎e-100) (Sup-
Sequences (MIPS ID; http://www.mips.biochem.mpg.de/) for A. plemental Table S1). All of the expression profiles for the four
thaliana genes corresponding to the rice FL-cDNA obtained were
searched by means of KOME. Arabidopsis FL-cDNAs corresponding
replications are available as GSE661, including GSM9853,
to the MIPS IDs of Arabidopsis genes obtained were searched using GSM9854, GSM9855 and GSM9856, on GPL477 in the NCBI
via the data from the RIKEN Arabidopsis Genome Encyclopedia GEO database. The upregulated genes included gene homologs
(RARGE; http://rarge.gsc.riken.go.jp/). From the 5⬘-end sequences already reported as responding to ABA (see “hormone, ABA”
of the FL-cDNAs of Arabidopsis (http://cdna01.dna.affrc.go.jp/ category in Supplemental Table S1) (21). Four clones [gene
cDNA/), the promoter sequences were obtained by comparison with homologs for aldose-reductase-related protein (AK066733),
the Arabidopsis genomic sequences on RARGE. We compared the glucose and ribitol dehydrogenase (AK110652), lipid transfer

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numbers of cis-elements between genes that respond to ABA or GA protein (AK062506), and the gene for globulin-like protein
in rice and corresponding genes in Arabidopsis and characterized the
(AK105347)] have been reported as responding not only to
types of cis-element in each plant. To characterize cis-elements in
each species, we divided the total number of each type of cis-element ABA but also to GA (34). Gene homologs for lipid transfer
in each species by the number of genes in each species. We then protein are not only ABA inducible but also salt and salicylic
compared the numbers of each type of cis-element per gene between acid inducible (7). Furthermore, it has been reported that, for
the two kinds of species. genes in the “storage protein” and “stress” categories in Sup-
plemental Table S1, there is cross talk between responses to
Quantitative Real-Time PCR such factors as low-temperature stress and ABA response (20).
Two micrograms of mRNA from each callus was denatured at The levels of expression of these genes verified the accuracy of
65°C for 5 min and then transferred to a bath at 37°C and incubated the experimental conditions. The gene for PBZ1 (AK071613;
for 5 min. At the same time, the first-strand reaction mix containing “defense” category in Supplemental Table S1), which was
mouse reverse transcriptase and d(T)18 primer (Amersham Phar- upregulated in our experiments, has been reported as a patho-
macia, Tokyo, Japan) was held at 37°C for 5 min. The RNA gen-related-protein gene (23). Upregulation of genes and gene
solution was transferred to the first-strand reaction mix for syn- homologs for development, transcriptional factors, and gener-
thesis of cDNA and incubated at 37°C for 60 min. Specific primers ation and differentiation in Supplemental Table S1 was also
for real-time PCR were designed to work under the same experi-
mental conditions (95°C for 10 min followed by 45 cycles of 95°C
detected with ABA treatment, although there were few com-
for 1 min and 60°C for 30 s), generating products of about 200–250 pared with the GA-responsive genes (described later). This
bp at the 3⬘-untranslated region (3⬘-UTR) of each expressed result suggests that the gene expression profiles in ABA-treated
sequence tag (EST) clone on the rice 8,987-cDNA microarray (34). callus look very similar to that in cells in the dormancy
Genes encoding heat shock protein (AU062924) and polyubiquitin process. Downregulated genes included those functionally
(AU108666, D22109) were tested as references for the hormonally annotated as responding to ABA (“hormone, ABA” category
treated and untreated samples. We chose the polyubiquitin gene in Supplemental Table S1) (38). Also, downregulation of a
(AU108666) for the standard lines because it was amplified by gene homolog for auxin response related to gravitropism
PCR faster than the other two genes were. Quantitative RT-PCR
(AK101504) was detected with ABA treatment (“hormone,
was performed with an iCycler (Bio-Rad, Tsukuba, Japan) and
SYBR Green reagent (Qiagen). For each reaction, standard lines others” category in Supplemental Table S1). Genes and
for both the treated and untreated samples were made by six gene homologs that were related to cell division and differen-
fivefold serial dilutions. The relative amounts of the target gene tiation of plant cells and that had not previously been reported
were calculated by comparison with standard lines of the poly- as ABA responsive, such as senescence-associated protein
ubiquitin gene (AU108666). (AK061848), root cap protein (AK108077), OsNAC3
(AK073667), and MADS-box-like protein (AK111859), were
RESULTS AND DISCUSSION downregulated by ABA treatment (“development”, “genera-
Genes with Altered Expression Levels in Callus Cultured tion, differentiation”, and “transcriptional factor” categories in
with ABA for 3 days Supplemental Table S1). Downregulation of these genes by
ABA treatment, which demonstrated seed dormancy, are con-
The standard deviation (SD) of the [log2(average ratio)] of sistent with the fact that plant cells are at a decreased level of
four expression replications of the experiment was 0.270 after growth during seed dormancy. Fourteen defense-related genes
the removal of flagged data, and all the selected genes in and gene homologs were identified as belonging to distinct
Supplemental Table S1 (available at the Physiological Genom- gene groups among those genes downregulated by ABA treat-
ics web site)1 were contained within the area of the median ment that demonstrated seed dormancy (“defense” category in
(0.00) ⫾3SD over a normal distribution. The changes in gene Supplemental Table S1). Eighty genes with unknown functions
were upregulated by ABA treatment, and 58 were downregu-
1
The Supplementary Material for this article (Supplemental Tables S1
lated by ABA treatment. These unknown genes were newly
through S10) is available online at http://physiolgenomics.physiology.org/ classified as ABA-responsive genes (“unclassified” category in
cgi/content/full/00201.2003/DC1. Supplemental Table S1).
Physiol Genomics • VOL 17 • www.physiolgenomics.org
90 PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE

We compared ABA response genes in plants among our OsDREB1B (AK062422; a drought-inducible gene) and a gene
result and other results of comprehensive expression analysis homolog for osmotic-inducible ankyrin kinase (AK100268),
(12, 29, 33) using MIPS code and accession ID (Table 1). were upregulated by GA treatment (“stress” category in Sup-
Among them, 200 (this report), 1,401 (12), 43 (29), and 493 plemental Table S2). Such genes had not previously been
(33) genes are candidates for ABA-inducible genes, respec- reported as GA responsive, except for metallothionein-related
tively. These inconsistencies of genes among these results may gene homologs (AK062653, AK103445) (38). Some of the
be attributed to the biological differences among plant species downregulated genes included had already been functionally
used, organ/tissues, ABA treatment condition, their response to annotated as responsive to GA (38) (“hormone, GA” and
ABA, and/or detection methodology. “storage protein” categories in Supplemental Table S2). These
genes included homologs of genes for hydrophobic LEA-like
Genes with Altered Expression Levels in Callus Cultured protein (AK102039) and RAB24 (AK102982), which was
with GA for 3 days reported as not only GA inducible but also antagonistically
ABA inducible (upregulated) (38). Eight genes related to
The SD of the [log2(average ratio)] of four replications of development had not previously been reported as GA respon-
the experiment was 0.311 after removal of flagged data, and all sive (“development” category in Supplemental Table S2). The
the selected genes in Supplemental Table S2 were contained in gene homolog for calcium-binding protein, CaBP1, in barley
the area of the median (0.00) ⫾3SD over a normal distribution.

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kernels (AK063625), which carries a single calcium-binding
The changes in gene expression were contained within the area EF-hand motif and one transmembrane helix and is upregu-
of the median (0.00) ⫾3SD over a normal distribution. Of the lated by endogenous ABA (15), was downregulated by our GA
301 genes selected, 206 were upregulated and 95 were down- treatment. The gene homolog of CaBP1 has shown that cal-
regulated; altered expression levels were identified by expres- cium-binding protein carrying a single EF-hand motif has
sion profile from analysis of the four oligonucleotide arrays in Ca2⫹-binding activity (6). Also, the gene homolog of CaBP1
two-dye swap experiments. Of these genes, 103 had functional was upregulated by our ABA treatment in two of the four
annotations in a BLASTN search on NCBI (⬎e-100) (Supple- replicated experiments (data not shown in Supplemental Table
mental Table S2). All the expression profiles for the analysis of S1; see deposited data for GSM9853 and GSM9854 of
the four oligonucleotide arrays in dye swap experiments were GSL661 in GEO at NCBI). The data from the other two
available as GSE661, including GSM9857, GSM9858, replications (data not shown in Supplemental Table S1; see
GSM9859, and GSM9860 on GPL477 in GEO at NCBI GSM9855 and GSM9856 of GSL661) on the DNA spotting on
(http://www.ncbi.nlm.nih.gov/geo/). The upregulated genes our microarray of the gene homolog were removed as flagged
included genes reported as responding to GA (“hormone, GA” data of saturated signal intensity. The gene for CaBP1 of barley
category in Supplemental Table S2) (38). Also, upregulation of was continuously detected in the vascular tissues in barley
genes for other hormone responses, including auxin-responsive kernels during development (15), and the gene homolog was
genes related to response to light (AK059838) and gravitro- upregulated by our ABA treatment and downregulated by our
pism (AK101191), was detected after GA treatment (“hor- GA treatment. These facts suggest that the gene homolog’s
mone, others” category in Supplemental Table S2). Thirty-one product might play key roles as a calcium receptor in highly
genes for development, cell division, generation, and differen- secretory organs during kernel development. In contrast, the
tiation in Supplemental Table S2 that had previously not been gene for calmodulin-binding protein, glutamate decarboxylase
reported as GA responsive were identified as being upregu- (AK061977; OsGAD1), which is expressed in various tissues,
lated. Such cell-division-related genes for plant growth were including maturing seeds (1), was not only downregulated by
not detected by ABA treatment. Twenty defense-related genes our GA treatment but also upregulated by our ABA treatment
were identified as belonging to distinct functional categories (Supplemental Table S1). We can speculate that the relation-
among those genes upregulated by GA treatment (“defense” ship of this gene’s activity to seed formation is demonstrated
category in Supplemental Table S2). The ABA and GA treat- by its upregulation by ABA treatment and downregulation by
ments revealed genes responsive to various physiological GA treatment. Some genes for stress response, such as gene
events in addition to their already known response to ABA or homologs responsive to low-temperature stress (AK070872,
GA. In particular, many genes for defense-related proteins AK073109) and genes responsive to submergence stress
were responsive to ABA and GA treatment. In expression (AK058296), which had not previously been reported as GA
analysis using the same oligonucleotide array, genes for de- responsive, were downregulated by GA (“stress” category in
fense-related proteins, including peroxidase, were also upregu- Supplemental Table S2). A gene (OsNAC4; AK073848) and
lated in germinating seeds (H. Yamada, NIAS, personal com- gene homologs (OsNAC6; AK068606, AK107746) of the NAC
munication). The detection of many kinds of genes for defense- family, related to development of plant tissue, were downregu-
related proteins suggests more strongly than our other report lated by our GA treatment (“transcriptional factor” category in
(38) that the ABA and GA response pathways have cross talk Supplemental Table S2). The NAC family is a group of
with pathogen-related pathways in rice callus. Besides, sali- transcriptional factors expressed in specific tissues such as
cylic acid (SA), a hormone known to mediate disease response, mature leaf (OsNAC3, 4, 6, 7, and 8), stem (OsNAC4, 6, 7, and
has recently been shown to positively or negatively regulate 8), root (OsNAC4, 5, 6, 7, and 8), embryos after pollination
cell enlargement and division (35), two physiological pro- (OsNAC5, 6, 7, and 8), and callus (OsNAC4, 5, 6, 7, and 8)
cesses known to be controlled by GA. Therefore, expression (17). However, there has been no report of the induction of
profiles of defense-related genes in ABA and GA treatment expression of these genes by GA treatment in tissues at the
callus show that may revealed the cross talk of SA, GA and germination stage. Other homologs of the OsNAC family,
ABA. Genes for stress response, such as the ABA-independent including OsNAC3, 5, 7, and 8 on our array, were expressed at
Physiol Genomics • VOL 17 • www.physiolgenomics.org
Table 1. Comparison of identified ABA-inducible genes in rice with those of Arabidopsis and other report of rice
Putative Gene Ratio* Accession No.‡
Average Identification of Induction
Clone Name Accession No. Ratio Putative Gene Identification of Rice Gene Category MIPS Code Arabidopsis Gene 1h 2h 5h 10 h 24 h Factor† EST FL-cDNA

Upregulated
J033072B12 AK073858 3.3 NADP-malic enzyme (Aloe arborescens) Development At2g19900 Malate oxidoreductase 2.4 3.2 4.4 9.3 5.3
(malic enzyme)
J023101G03 AK071637 3.2 Protein phosphatase 2C Development At5g24940 Protein phosphatase BP432943 AK071637
(Mesembryanthemum crystallinum) 2C-like protein
006-210-H01 AK061224 2.5 Raffinose synthase, Rfs (Cucumis sativus) Development At5g40390 Glycosyl hydrolase 5.2 2.5 1.5 1.3 1.9
family 36
J023078G01 AK100306 1.9 Sucrose synthase type 2 (Triticum Development At3g43190 Sucrose synthase-like ⫺4.4
aestivum) protein
002-169-E03 AK110652 9.4 Glucose and ribitol dehydrogenase Hormone, At1g54870 Dormancy related 1.4 1.6 1.9 1.7 2.6
homolog (Hordeum vulgare) ABA protein, putative
001-117-H05 AK063578 2.8 ABA-responsive protein (H. vulgare) Hormone, At5g13200 ABA-responsive 4.1 BP432968 AK063578
ABA protein-like
001-124-F05 AK105433 2.3 Heat shock protein, HSP101 (Oryza Stress At1g74310 Heat shock protein 101 3.0
sativa) (HSP101)
001-119-F01 AK063685 2.9 Homeodomain leucine zipper protein, Transcriptional At2g46680 Homeodomain 6.8 7.5 8.8 18.8 13.9 27.7
Oshox6 (O. sativa) factor transcription factor
(ATHB-7)
001-118-H06 AK063645 6.6 Unknown Unclassified At1g01470 Hypothetical protein 5.8 7.2 4.0 5.1 2.7 4.6
002-140-G05 AK108233 6.1 Unknown Unclassified At2g22170 Unknown protein ⫺5.8

Physiol Genomics • VOL


002-153-F10 AK108988 4.7 Unknown Unclassified At3g48530 Putative transcription 36.0

17 •
factor X2 (O. sativa)
J023012K18 AK069274 4.1 Unknown Unclassified At1g07430 Protein phosphatase 2C 115.0
(PP2C)
002-169-F02 AK110661 3.5 Unknown Unclassified At1g03790 Unknown protein 25.0
containing CCCH-
type zinc finger
J013146J07 AK068272 2.9 Unknown Unclassified At5g59220 Protein phosphatase 2C 36.6
(PP2C)
002-175-E10 AK111075 2.8 Unknown Unclassified At1g27300 Hypothetical protein 3.1
J023037K22 AK069960 2.7 Unknown Unclassified At5g54160 O-methyltransferase 1 BP433009 AK064768

www.physiolgenomics.org
001-120-E03 AK063729 2.7 Unknown Unclassified At3g18280 Lipid transfer protein, 1.0 1.2 1.5 2.5 3.4
putative
002-124-C12 AK107138 2.2 Unknown Unclassified At5g14180 Putative protein 1.6 2.7 3.9 7.1 6.4 ⫺6.8
PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE

Downregulated
002-114-G10 AK106723 0.4 ␣-Amylase/subtilisin inhibitor, BASI (H. Hormone, At1g73260 Putative trypsin 1.5 2.4 9.7 11.9 7.0 3.7
vulgare) ABA inhibitor
002-108-G04 AK064395 0.5 IAA (anxin indole-3-acetic acid)-glu Hormone, At2g43820 Putative 1.0 1.8 2.1 3.1 3.3
synthetase, iaglu (Zea mays) others glucosyltransferase
J033052E11 AK073667 0.4 OsNAC3 protein (O. sativa) Transcriptional At1g77450 GRAB1-like protein 18.1
factor
002-111-B07 AK064485 0.4 Unknown Unclassified At3g02040 Hypothetical protein 3.1
001-123-G04 AK063959 0.3 Unknown Unclassified At2g38290 Ammonium transporter 17.0
AtAMT2
MIPS Code indicates gene identification of the sequence in the Munich Information Center for Protein Sequences (MIPS). *cDNA microarray data for a set of transcripts previously reported as ABA
responsive (33) and modified here were compared with our expression data. †Expression profile of ABA-responsive gene previously reported (12) and modified here was compared with our data. ‡cDNA
microarray data for a set of transcripts previously reported as ABA responsive (29) and modified here were compared with our data. Details of all experimental conditions refer to each report (12, 29, 33).
See legend to Supplemental Table S1 for explanation of other column names. ABA, abscisic acid; FL-cDNA, full-length complementary DNA; EST, expressed sequence tag.
91

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92 PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE

almost the same level (average ratio ⫽ 1.0384) in GA-treated defense-related genes and gene homologs by ABA and GA
and untreated callus. (See deposited data, GSE661 including treatment suggests that the plant seed does not require high
GSM9857 through GSM9860, at the NCBI GEO database.) levels of protection from threats such as pathogens in the
The gene homolog (AK063685) for the homeodomain leucine external environment, but that at germination the plant is more
zipper protein family, which has a role as a developmental vulnerable and the defense-related genes are therefore upregu-
regulator, was newly found to be downregulated by GA. We lated. The fact that a gene for MADS-box-like protein
found 132 genes with unknown functions that were upregu- (AK111859), which functions in the construction of the repro-
lated by GA treatment and 66 that were downregulated. These ductive organs (30), was regulated antagonistically by ABA
unknown genes were newly classified as GA responsive. and GA suggests that the gene might have a new function
We performed a comprehensive analysis of gene expression related to seed dormancy and germination. Forty-two clones of
using callus tissue treated with ABA or GA. Genes for em- genes with unknown functions were newly assigned functions
bryogenesis, germination, seed dormancy, and grain filling as genes antagonistically responsive to ABA and GA treat-
were included among those with altered expression levels. The ment. Fifteen genes were reported as representing genes re-
fact that the experiments identified many already known ABA- sponsive to both hormones in our previous study, which used
and GA-responsive genes in other tissues shows that callus the rice 8,987-cDNA microarray and quantitative RT-PCR
tissue can be mimic the mechanisms of germination and (38). In contrast, we detected 66 genes responsive to both

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dormancy. The high number of responsive genes detected culture with GA and culture with ABA, which is about 4 times
showed that this analysis, which used our oligonucleotide as many as in previous reports (38), from an analysis of our
array, was more efficient than those used in our other report oligonucleotide array. We hope that our results will add to the
(38) for genome-wide functional analysis of rice. currently incomplete research data on the interaction between
the ABA and GA responses in plant dormancy and germina-
Genes Responsive to Both Culture with GA and Culture tion.
with ABA
Specification of Cis-Elements of ABA- and
To elucidate the interaction between ABA and GA, we GA-Responsive Genes
selected 68 genes that had shown a response to both hormone
treatments (Supplemental Table S3). Sixty-six of the 68 genes To elucidate the mechanism of transcriptional regulation of
showed antagonistic responses to the 2 hormones. Twenty-five the hormone-response systems of plants, we analyzed the
of these genes had functional annotations in a BLASTN search cis-elements of genes that were responsive to our hormone
at NCBI (⬎e-100). Two genes were either upregulated or treatment. We assigned our FL-cDNAs on the rice genome
downregulated by both hormones. Of the 66 antagonistically constructed by bacterial artificial chromosome (BAC) contigs
regulated genes, 34 were upregulated by ABA and downregu- from the results of the TIGR Rice Genome Project (http://
lated by GA, and 32 were downregulated by ABA and upregu- www.tigr.org/tdb/e2k1/osa1/BACmapping/description.shtml)
lated by GA. The former 34 genes included LEA family genes and searched the cis-elements up to 1,000 bp upstream from
(AK102039, AK102982) previously reported as antagonisti- the cDNA from the 5⬘ terminus in the rice genomic sequence.
cally responding to both hormones (“hormone” category in The results of this cis-element analysis of all the clones on our
Supplemental Table S3) (38). Although there have been no oligonucleotide array are available on the KOME web site. The
previous reports of a gene for cysteine protease inhibitor numbers of all the types of cis-elements of genes responsive to
(AK105278) having antagonistic responses to the two hor- our treatments are shown in Supplemental Tables S4 and S5.
mones, our result was not surprising, in that one would expect The results of comparison 1 (see MATERIALS AND METHODS)
ABA to promote such a gene for inhibiting hydrolysis of among 76 genes upregulated by ABA, 61 genes downregulated
storage protein and GA to inhibit it. The pattern of expression by GA, and 34 genes both upregulated by ABA and down-
of a gene for cysteine proteinase (AK106508), which was regulated by GA are summarized in Table 2A. Two kinds of
downregulated by ABA and upregulated by GA, verifies this cis-element for light response (IBOX, BOXIIPCCHS) and
result. Genes and gene homologs for stress response, including seven for protein storage response were remarkably rich in the
response to low temperature (AK073109, AK070872), have upstream regions of genes upregulated by ABA and downregu-
cross talk with the ABA-responsive genes (20) that were lated by GA. Cis-elements for two kinds of storage protein
detected in both our treatments (“stress” category in Supple- (RAV1BAT, CANBNNAPA) were specified as elements of
mental Table S3). Gene homologs for homocysteine S-meth- genes upregulated only by ABA in the comparison. Cis-
yltransferase-4 (AK073362) and homeodomain leucine zipper elements for defense (SEBFCONSSTPR10A), ethylene
protein (AK063685), which have various known functions, (ERELEE4), and development (ACGTCBOX) responses were
including roles as developmental regulators, were assigned specified as elements of genes downregulated only by GA in
new functions related to the ABA and GA response, which the comparison. Cis-elements for dehydration stress response
modeled seed dormancy and germination. The 32 genes in (MYCATRD22) and light response (TBOXATGAPB) were
Supplemental Table S3 that were downregulated by ABA and specified as elements of both the gene group upregulated only
upregulated by GA included 8 clones of defense-related genes by ABA and the group downregulated only by GA. The results
and gene homologs that had not previously been reported as of comparison 2 (see MATERIALS AND METHODS) among 173
antagonistically responsive (38) (“defense” category in Sup- genes upregulated by GA, 57 genes downregulated by ABA,
plemental Table S3) and gene homologs for development and and 32 genes both upregulated by GA and downregulated by
stress (“development” and “stress” categories in Supplemental ABA are summarized in Table 2B. Two kinds of cis-element
Table S3). Detection of such changes in the expression of these for amylase (AMYBOX1, TATCCAOSAMY) were rich in the
Physiol Genomics • VOL 17 • www.physiolgenomics.org
PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE 93
Table 2. Specification of cis-elements of ABA- and GA-responsive genes
Gene Group Category Cis-elements Specificities

A: Comparison of cis-element numbers among genes upregulated by ABA, downregulated by GA, and both upregulated by ABA and downregulated by GA
Upregulated by ABA, downregulated by GA Development MYBPZM
Light IBOX, BOXIIPCCHS
Other hormone CATATGGMSAUR, MARTBOX
Protein storage RYREPEATVFLEB4, ACGTABREMOTIFA2OSEM,
DPBFCOREDCDC3, CACGTGMOTIF,
RYREPEATLEGUMINBOX, TATABOX3,
RYREPEATGMGY2
Downregulated by GA Defense SEBFCONSSTPR10A
Development ACGTCBOX
Light TBOXATGAPB
Other hormone ERELEE4
Stress (dehydration) MYCATRD22
Upregulated by ABA Light TBOXATGAPB
Storage protein RAV1BAT, CANBNNAPA

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Stress (dehydration) MYCATRD22
B: Comparison of cis-element numbers among genes upregulated by GA, downregulated by ABA, and both upregulated by GA and downregulated by ABA
Upregulated by GA, downregulated by ABA Amylase AMYBOX1, TATCCAOSAMY
Light IBOX, ⫺10PEHVPSBD
Stress (various) MYBST1
Upregulated by GA Defense SEBFCONSSTPR10A
Light BOXIINTPATPB
Stress (various) MYBPLANT, TATABOXOSPAL
Downregulated by ABA Defense SEBFCONSSTPR10A
Development ACGTABOX
Stress (various) LTRECOREATCOR15, PALBOXAPC
“Cis-elements specificities” indicates cis-elements that are rich in the upstream regions of genes in each group. Details of each cis-element can be found in
the PLACE database (http://www.dna.affrc.go.jp/htdocs/PLACE/). GA, gibberellin.

upstream regions of genes that were both upregulated by GA external environment during germination, or that germination
and downregulated by ABA. Genes downregulated by ABA is in fact a phenomenon of self-stress. In addition, these results
and genes upregulated by GA were rich in cis-elements for suggest that plant cells are resistant to receive stress from the
stress response (MYBPLANT, TATABOXOSPAL, upregu- external environment during seed dormancy. We compared the
lated by GA; LTRECOREATCOR15, PALBOXAPC, down- cis-elements for tissue-specific response between parts A and B
regulated by ABA) in the upstream regions. Cis-element for of Table 2. There are nine kinds of cis-element for seed protein
the defense response (SEBFCONSSTPR10A) was specified as storage in Table 2A. Seven kinds of element for protein storage
an element common to both the gene group upregulated by GA were included in the gene group both upregulated by ABA and
and the group downregulated by ABA. These results suggest downregulated by GA, demonstrating that this group is related
that antagonistic-response genes regulated by ABA treatment to seed dormancy. In contrast, two elements (AMYBOX1,
(Table 2A) or GA treatment (Table 2B) have cis-elements such TATCCAOSAMY) for seed germination response were in-
as RYREPEATVFLEB4 (“protein storage” category in Table cluded in the gene group both upregulated by GA and down-
2A) and AMYBOX1 (“amylase” category in Table 2B), which regulated by ABA (Table 2B) and were thus related to seed
are related to seed dormancy and germination, respectively. In
germination. The variety of cis-elements for protein storage in
contrast, genes for response to only one of the two hormones
the gene group both upregulated by ABA and downregulated
are rich in elements such as MYCATRD22 [“stress (dehydra-
by GA (Table 2A), which mimics seed dormancy, is larger than
tion)” category in Table 2A] and SEBFCONSSTPR10A (“de-
fense” category in Table 2B), which promote seed dormancy those for amylase in the gene group both upregulated by GA
and germination, respectively. and downregulated by ABA (Table 2B), which mimics germi-
Cis-elements for stress response were richer in the gene nation. We do not know whether the cis-elements of the latter
groups of Table 2B than those of Table 2A. There are five group have multiple functions or whether they are poorly
elements for stress-response factor (MYBST1, MYBPLANT, represented on the PLACE database.
TATABOXOSPAL, LTRECOREATCOR15, and PALBOX- In promoter sequences search of the ABA response genes
APC) in Table 2B. MYBST1, for responding to various obtained, we detected that several genes (AK064966,
stresses, was included in the gene group both upregulated by AK073100, AK073380, AK073777, AK073833, AK102307,
GA and downregulated by ABA, which demonstrates that it is AK103170, AK105316, AK106508, AK108159, AK110259,
related to stress response in seed germination. In contrast, one and AK110912 in Supplemental Table S4) did not contain any
element for dehydration stress response (MYCATRD22) was ABA-responsive element in their promoters as well as other
rich in genes upregulated by ABA and genes downregulated by report (29). These results suggest the existence of novel cis-
GA (“stress” category in Table 2A). We speculate that plant acting elements involved in ABA-inducible gene expression in
cells are particularly susceptible to receive stress from the their promoters.
Physiol Genomics • VOL 17 • www.physiolgenomics.org
94 PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE

Phenotypes of Transposon Insertion Mutants in legumes at the onset of germination and during the early
stage of nodule development (3). The phenotypes of dwarfism,
To elucidate the functions of the genes responsive to our brittleness, and lethality indicate growth delay and suggest that
treatments, we investigated the phenotypes of mutants in which
disruption of this gene led to reduced protein synthesis for
the rice retrotransposon Tos17 was inserted in the regions of
plant growth using nitrogen because of disruption of nitrogen
these responsive genes with the aid of a Tos17 mutant panel
metabolism pathways. Because, unlike legumes, rice does not
database (http://tos.nias.affrc.go.jp/). Tos17, which is highly
activated by tissue culture, has been used for insertion mu- form root nodules, we speculate that this gene homolog may
tagenesis of rice (24). The Tos17 mutant panel database en- play a role in nitrogen metabolism with regard to plant growth
ables one to link flanking sequences with phenotype informa- dependent on ABA and GA signal transduction. The phenotype
tion by using the BLAST program. With this database, one can of the insertion mutant of the gene homolog for protein
perform gene function analysis by computer and reverse ge- phosphatase (AK068272) of Lotus japonicus, which has a
netics. We obtained 12 insertion mutants from the 200 ABA- function in nodules that are in the process of initiating nitrogen
responsive genes (Table 3). Gene homologs for ankyrin kinase fixation (16), showed sterility. Loss of function of the gene
(AK100268), protein phosphatase type 2C (AK068272), and homolog in response to Tos17 insertion might reduce protein
zinc transporter protein (AK105258) had functional annota- storage during seed formation by disruption of a nitrogen
tions in a BLASTX search on NCBI (identities ⬎40%) and in metabolism pathway. We speculate that the gene homolog may

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published reports. The phenotypes of the insertion mutants of function partly in nitrogen metabolism with respect to seed
the gene homolog for ankyrin kinase (AK100268) of alfalfa, formation dependent on ABA signal transduction. The pheno-
induced by not only ABA but also by GA, showed dwarfism, types of insertion mutants of the gene homolog for zinc
brittleness, and lethality. The gene, containing an NH2-termi- transporter protein (AK105258), which plays a housekeeping
nal region with an ankyrin domain, plays a role in signal role in zinc metabolism of soybean nodules (26), showed
transduction in various developmental processes, particularly abnormalities, kernel with a white base, of seed formation in

Table 3. Results of a phenotype search of insertion mutants of ABA-responsive genes, using Tos17
BLASTN BLASTX
Average
Clone Name Accession No. Ratio Tos17 ID Phenotype Putative Gene ID Accession No. Putative Gene ID Accession No.

002-112-G01 AK064587 4.82 NC2670_0_404_1A No comment Clone 26360 AY086623.1 Putative eukaryotic peptide AY034963.1
(Arabidopsis chain release factor
thaliana) subunit 1 (A. thaliana)
002-112-G01 AK064587 4.82 H0732_1_102_1A Dwarf Clone 26360 AY086623.1 Putative eukaryotic peptide AY034963.1
(A. thaliana) chain release factor
subunit 1 (A. thaliana)
J033082H02 AK102075 3.14 NE8550_0_105_1A Dwarf Unknown AT5g23390/T32G24_2 AY090230.1
mRNA (A. thaliana)
J013146J07 AK068272 2.91 T09854T Sterility Unknown Nodule-enhanced protein AF092431.1
phosphatase type 2C,
NPP2C1 (Lotus
japonicus)
002-105-E08 AK064271 2.59 T14722T Sterility Unknown Heat stress transcription X67601.1
factor 30, Lp-hsf30
(Lycopersicon
peruvianum)
001-114-G07 AK105258 2.35 ND5061_0_103_1A No comment Unknown Zinc transporter protein, AY029321.1
ZIP1 (Glycine max)
001-114-G07 AK105258 2.35 NE1520_0_101_1A White-based Unknown Zinc transporter protein, AY029321.1
rice kernel ZIP1 (G. max)
006-303-C01 AK109407 0.47 NC0021_0_702_1A Viviparous, Unknown AT3g48690/T8P19_200 AY064980.1
Dwarf, mRNA (A. thaliana)
Chlorina
002-173-G04 AK110936 0.39 NF5045_0_405_1A Growth delay, Unknown Benzoic acid carboxyl AF198492.1
Zebra leaf, methyltransferase,
Dwarf BAMT (Antirrhinum
majus)
J023065A22 AK100268 0.36 NC0584_0_403_1A No comment Ankyrin-kinase AF458699.1 Ankyrin-kinase (M. AF458699.1
(Medicago truncatula)
truncatula)
J023065A22 AK100268 0.36 T01783T Dwarf, Brittle Ankyrin-kinase AF458699.1 Ankyrin-kinase (M. AF458699.1
(M. truncatula) truncatula)
J023065A22 AK100268 0.36 T12791T Lethal, Dwarf, Ankyrin-kinase AF458699.1 Ankyrin-kinase (M. AF458699.1
Low (M. truncatula) truncatula)
germination
rate
“Tos17 ID” indicates the original identification number of the flanking sequence submitted to the Rice Tos17 Insertion Mutant Database. “Phenotype” indicates the
original comment on the picture of the mutant submitted to the Rice Tos17 Insertion Mutant Database. See caption to Supplemental Table S1 for explanation of other
column names.

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PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE 95
rice. Although of course rice does not form root nodules, the and cysteine synthase have an effect on tissue-specific devel-
effects of zinc deficiency on nitrogen metabolism of meristem- opment (seed formation) and may function farther downstream
atic tissues with regard to protein synthesis in rice have been in the nitrogen metabolism pathway than the ankyrin kinase.
reported (19). The phenotypes suggest that loss of function of However, because these two genes are regulated by different
the gene homolog of zinc transporter protein in response to hormones, and one gene homolog (for cysteine synthase) is
Tos17 insertion might suppress protein synthesis. We speculate related to the sulfate metabolism pathway, the nitrogen metab-
that this gene homolog’s product may function farther up- olism pathway based on ABA metabolism might be different
stream in the nitrogen metabolism pathway than does protein from that based on GA metabolism, and the two pathways
phosphatase with regard to protein synthesis dependent on of nitrogen metabolism may interact on ankyrin kinase (for
ABA signal transduction. We obtained 29 kinds of insertion ABA, ankyrin kinase 3 protein phosphatase; for GA, ankyrin-
mutant from the 301 GA-responsive genes (Table 4). Gene kinase 3 cysteine synthase). Also, the zinc transporter, which
homologs for ␤-mannosidase (AK068499), cysteine synthase has a transmembrane domain, has an effect on tissue-specific
(AK072457), ankyrin kinase (AK100268), and seven-trans- development (seed formation) and may function farther up-
membrane protein, Mlo8 (AK072733) had functional annota- stream in the nitrogen metabolism pathway than does protein
tions in a BLASTX search of NCBI and in published reports. phosphatase without interaction of ankyrin kinase with a trans-
The phenotypes of tomato containing insertion mutants of the membrane domain (zinc transporter 3 protein phosphatase).
gene homolog for ␤-mannosidase (AK068499), which is in-

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Second, the results in Table 4 suggest that GA signal
volved in the mobilization of galactomannans in the cell wall transduction interacts with the cell wall metabolic pathway
of the lateral endosperm in the early stages after germination through its effects on mannosidase and invertase. We can
(25), showed growth delay. Loss of function of the gene speculate that mannosidase is relevant to polysaccharide me-
homolog in response to Tos17 insertion in rice might reduce tabolism, which has a wide effect on plant growth, and may
the synthesis of proteins for plant growth by disrupting a function farther upstream in the cell wall metabolism pathway
carbohydrate metabolism pathway; accordingly, the pheno-
than invertase, which is relevant to disaccharide metabolism.
types included growth delay. We speculate that the gene
Third, the results in Table 4 suggest that GA signal transduc-
homolog may play a role in carbohydrate metabolism with
tion interacts with signal transduction of biotic stress through
regard to plant growth dependent on GA signal transduction.
Mlo8. We can speculate that other genes such as ankyrin-
The phenotype of rice carrying the insertion mutant of the gene
homolog for cysteine synthase (AK072457), which functions kinase (plant height) and cysteine synthase (sterile) down-
in the regulation of sulfur and nitrogen availability (28), stream of the protein of Mlo8 have a wide effect (plant height
included sterility. Loss of function of the gene homolog in and sterility) on plant growth. These results and speculations,
response to Tos17 insertion might reduce protein storage abil- derived from phenotype analysis by molecular simulation us-
ity during seed formation by disruption of a nitrogen metabo- ing genome-wide expression profiles and a huge mutant col-
lism pathway. We speculate that its protein product may lection, represent new knowledge of the systematic cataloguing
function in nitrogen and sulfur metabolism with regard to seed of ABA and GA responses in rice.
formation dependent on GA signal transduction. The pheno- However, the phenotypes of transposon insertion mutants in
types of alfalfa carrying an insertion mutant of the gene our data are preliminary data from the Tos17 mutant panel
homolog for ankyrin kinase (AK100268), induced not only by database. The studies are underway not only to analyze
GA but also by ABA in our experiments, included dwarfism, whether they are a homozygous line or not but also to analyze
brittleness, and lethality. We speculate that the gene functions consistent of the phenotype among different lines of the same
as described at phenotyping of ABA response genes, earlier. gene and to analyze whether the mutant phenotypes are rescu-
The phenotype of rice insertion mutant of the gene homolog for able.
Mlo8 (AK072733), which is involved in defense, response to
biotic stress, and leaf senescence (4), included dwarfism and Specification of Cis-Elements of ABA/GA-Responsive Genes
sterility. Loss of function of the gene homolog in response to in Rice Callus and Arabidopsis Genes Corresponding to
Tos17 insertion might cause suppression of the cell develop- Genes for ABA/GA Response of Rice Callus
ment in plant height and seed formation, because of reduced
stress response. To characterize the mechanism of transcriptional regulation
From these phenotypic results and published reports, we of the hormone response systems in rice, we analyzed cis-
suggest that ABA and GA signal transduction interacts with elements of genes of Arabidopsis corresponding to genes for
three signal-transduction pathways responsible for nutrient ABA response of rice and compared the types of cis-element in
metabolism, cell wall metabolism, and biotic-stress defense. each species. In genes with cis-element more than one element
First, the phenotypic results in Tables 3 and 4 suggest that in total number of each type of element, we showed cis-
ABA and GA signal transduction interacts with nutrient met- element profile of 116 (rice) and 94 (Arabidopsis) clones in
abolic pathways, especially in nitrogen metabolism, through Supplemental Tables S6 and S7, respectively. The results of
gene homologs for ankyrin kinase, protein phosphatase, zinc comparison between 116 clones responsive to ABA in rice and
transporter protein, and cysteine synthase. We can speculate 94 clones of corresponding Arabidopsis genes are summarized
that the gene homolog for ankyrin kinase, which has a trans- in Table 5. From all detected cis-elements, we selected cis-
membrane domain, has a wide effect on plant growth and may elements for determination of specific elements that were
function further upstream in the nitrogen metabolism pathway present in at least two genes in either species and specific
than the products of the other two genes (protein phosphatase, element were more than ⫾2-fold in the ratio of (rice/Arabi-
cysteine synthase). And we suggest that protein phosphatase dopsis). Also, we selected cis-elements for determination of
Physiol Genomics • VOL 17 • www.physiolgenomics.org
96 PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE

Table 4. Results of a phenotype search of insertion mutants of GA-responsive genes, using Tos17
BLASTN BLASTX
Accession Accession Accession
Clone Name No. Average Ratio Tos17 ID Phenotype Putative Gene ID No. Putative Gene ID No.

001-019-H08 AK058750 0.35 NC0238_0_407_1A No comment myo-Inositol AB012107.1 myo-Inositol phosphate synthase, AB012107.1
phosphate RINO1 (O. sativa)
synthase, RINO1
(Oryza sativa)
001-113-C06 AK063277 2.24 NF3020_0_104_1A No comment Unknown Unknown
001-200-B09 AK105614 2.64 NF4021_0_401_1A No comment Unknown PV72 (Cucurbita cv. Kurokawa AB006809.1
Amakuri)
002-112-E08 AK064577 0.37 T24162T No comment Unknown Unknown
002-112-E08 AK064577 0.37 T08819T Vivipary Unknown Unknown
J013000A05 AK064768 2.71 T11716T Dwarf Caffeic acid 3-O- AJ231133.1 Caffeic acid O-methyltransferase AF153825.1
methyltransferase (Festuca arundinacea)
(Saccharum
officinarum)
J013099F12 AK067444 2.62 T11806T Albino Cell wall invertase, AF030421.1 Cell wall invertase, IVR3 (T. AF030421.1
IVR3 (Triticum aestivum)

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aestivum)
J013099F12 AK067444 2.62 NE4523_0_501_1A Albino Cell wall invertase, AF030421.1 Cell wall invertase, IVR3 (T. AF030421.1
IVR3 (T. aestivum)
aestivum)
J013099F12 AK067444 2.62 NF1010_0_401_1A No comment Cell wall invertase, AF030421.1 Cell wall invertase, IVR3 (T. AF030421.1
IVR3 (T. aestivum)
aestivum)
J013099F12 AK067444 2.62 ND3069_0_103_1A No comment Cell wall invertase, AF030421.1 Cell wall invertase, IVR3 (T. AF030421.1
IVR3 (T. aestivum)
aestivum)
J013099F12 AK067444 2.62 T16055T Albino Cell wall invertase, AF030421.1 Cell wall invertase, IVR3 (T. AF030421.1
IVR3 (T. aestivum)
aestivum)
J013099F12 AK067444 2.62 T14840T Sterile Cell wall invertase, AF030421.1 Cell wall invertase, IVR3 (T. AF030421.1
IVR3 (T. aestivum)
aestivum)
J013099F12 AK067444 2.62 T13126T White belly rice Cell wall invertase, AF030421.1 Cell wall invertase, IVR3 (T. AF030421.1
kernel IVR3 (T. aestivum)
aestivum)
J013151A14 AK068457 2.57 NC0285_0_101_1A No comment Unknown At2g24280/F27D4.19 mRNA AY058177.1
(Arabidopsis thaliana)
J013151A14 AK068457 2.57 NC0347_0_103_1A Sterile Unknown At2g24280/F27D4.19 mRNA (A. AY058177.1
thaliana)
J013151A14 AK068457 2.57 NF8538_0_702_1A Sterile (fertility Unknown At2g24280/F27D4.19 mRNA (A. AY058177.1
10–25%) thaliana)
J013155M12 AK068499 0.14 T14065T No comment Unknown ␤-Mannosidase enzyme AF403444.1
(Lycopersicon esculentum)
J013155M12 AK068499 0.14 NF2766_0_106_1A Chlorina, Unknown ␤-Mannosidase enzyme (L. AF403444.1
Growth esculentum)
delay, sterile
J023028D15 AK069659 2.52 NF7016_0_103_1A No comment Unknown Putative male sterility 2 protein AY051075.1
(A. thaliana)
J023065A22 AK100268 2.25 NC0584_0_403_1A No comment Ankyrin-kinase AF458699.1 Ankyrin-kinase (M. truncatula) AF458699.1
(Medicago
truncatula)
J023065A22 AK100268 2.25 T01783T Dwarf, Brittle Ankyrin-kinase (M. AF458699.1 Ankyrin-kinase (M. truncatula) AF458699.1
truncatula)
J023065A22 AK100268 2.25 T12791T Lethal, Dwarf Ankyrin-kinase (M. AF458699.1 Ankyrin-kinase (M. truncatula) AF458699.1
truncatula)
J023096E14 AK071445 2.92 NF3013_0_732_1A No comment Unknown Unknown
J023123O18 AK072457 2.10 NC0263_0_103_1A Sterile Cysteine synthase, AF073698.1 Cysteine synthase, rcs4 (O. AF073698.1
rcs4 (O. sativa) sativa)
J023140L15 AK072727 2.24 NC0230_0_105_1A No comment Unknown At2g42580/F14N22.15 mRNA AF367321.1
(A. thaliana)
J023141N06 AK072733 2.87 T10727T No comment Seven AY029319.1 Seven transmembrane protein, AY029319.1
transmembrane M1o8 (Z. mays)
protein, M1o8
(Zea mays)
J023141N06 AK072733 2.87 T03437T No comment Seven AY029319.1 Seven transmembrane protein, AY029319.1
transmembrane M1o8 (Z. mays)
protein, M1o8
(Z. mays)
J023141N06 AK072733 2.87 ND8013_0_702_1A Dwarf, Sterile Seven AY029319.1 Seven transmembrane protein, AY029319.1
(fertility 50– transmembrane M1o8 (Z. mays)
70%) protein, M1o8
(Z. mays)
J033136K04 AK103697 2.39 T15150T No comment Unknown Chorismate synthase (Corydalis X63595.1
sempervirens)

See caption to Table 3 for explanation of column names.

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PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE 97
Table 5. Specification of cis-element of Arabidopsis and rice gene for ABA response
Specific or Common Rice (116 clones) Cis-element Arabidopsis (94 clones) Ratio Category

Rice specific 81 INTRONLOWER 17 3.86 3⬘ Intron-exon splice junctions


Rice specific 314 CGACGOSAMY3 62 4.1 Amylase
Rice specific 86 PALBOXAPC 10 6.97 Defense
Rice specific 131 MYBPZM 41 2.59 Development (pigmentation in floral organ)
Rice specific 70 HEXAMERATH4 13 4.36 Histone H4 promoter
Rice specific 44 ABREOSRAB21 0 ND Hormone: ABA
Rice specific 69 RAV1BAT 19 2.94 Hormone: ABA
Rice specific 59 ⫺10PEHVPSBD 0 ND Primary metabolism: light
Rice specific 113 ⫺300ELEMENT 0 ND Protein storage
Rice specific 69 RYREPEATGMGY2 19 2.94 Protein storage
Rice specific 97 RYREPEATLEGUMINBOX 31 2.54 Protein storage
Rice specific 171 RYREPEATBNNAPA 58 2.39 Protein storage
Rice specific 169 LTRECOREATCOR15 60 2.28 Stress: low temperature stress
Common 265 WBOXATNPR1 223 0.96 Defense
Common 305 DPBFCOREDCDC3 140 1.77 Hormone: ABA
Common 298 RAV1AAT 301 0.8 Hormone: ABA

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Common 1,454 DOFCOREZM 1,477 0.8 Hormone: GA
Common 947 GT1CONSENSUS 948 0.81 Primary metabolism: light
Common 938 GATABOX 899 0.85 Primary metabolism: light
Common 304 IBOXCORE 320 0.77 Primary metabolism: light
Common 260 TATABOX5 338 0.62 Primary metabolism: light
Common 200 INRNTPSADB 215 0.75 Primary metabolism: light
Common 160 CACGTGMOTIF 96 1.35 Primary metabolism: light
Common 240 POLASIG1 326 0.6 Primary metabolism: polyA
Common 162 POLASIG3 224 0.59 Primary metabolism: polyA
Common 1,004 GTGANTG10 682 1.19 Primary metabolism: tissue (late pollen)
Common 613 POLLEN1LELAT52 651 0.76 Primary metabolism: tissue (pollen)
Common 547 ROOTMOTIFTAPOX1 700 0.63 Primary metabolism: tissue (root)
Common 242 TAAAGSTKST1 329 0.6 Primary metabolism: tissue (guard cell)
Common 1,222 EBOXBNNAPA 812 1.22 Protein storage
Common 1,119 CAATBOX1 1,128 0.8 Protein storage
Common 242 SEF4MOTIFGM7S 249 0.79 Protein storage
Common 251 CCAATBOX1 227 0.9 Stress: heat shock
Common 352 MYBCORE 213 1.34 Stress: water stress
Common 199 MYBST1 146 1.11 Unclassified
Arabidopsis specific 48 AMYBOX1 83 0.47 Amylase
Arabidopsis specific 44 TATABOXOSPAL 73 0.49 Defense
Arabidopsis specific 34 MYBGAHV 59 0.47 Hormone: GA
Arabidopsis specific 57 TATABOX4 103 0.45 Primary metabolism: light
Arabidopsis specific 37 TATABOX3 74 0.41 Primary metabolism: light
Arabidopsis specific 0 C8GCARGAT 208 ND Primary metabolism: MADS-domain
Arabidopsis specific 30 TATAPVTRNALEU 51 0.48 Primary metabolism: reinitiation
Arabidopsis specific 118 MARTBOX 192 0.5 Primary metabolism: scaffold
Arabidopsis specific 0 MYCCONSENSUSAT 812 ND Stress: dehydration
Arabidopsis specific 0 ACGTATERD1 576 ND Stress: dehydration
Arabidopsis specific 0 ABRELATERD1 201 ND Stress: dehydration
Arabidopsis specific 0 MYB1AT 181 ND Stress: dehydration
Arabidopsis specific 0 MYB2CONSENSUSAT 96 ND Stress: dehydration
Arabidopsis specific 0 MYCATERD1 61 ND Stress: dehydration
The “Specific or common” column shows the cis-elements that are rice specific, Arabidopsis specific, or common to both species. “Rice” indicates the total
number of cis-elements of 116 clones responsive to ABA treatment within 1,000 bp upstream of the 5⬘ region of each gene in rice. Details of all cis-elements
refer to the PLACE database (http://www.dna.affrc.go.jp/htdocs/PLACE/). Italic characters show cis-elements that exist only in Arabidopsis or rice.
“Arabidopsis” indicates the number of cis-elements in 94 clones of Arabidopsis corresponding to rice genes for ABA response. The data were obtained from
RARGE (http://rarge.gsc.riken.go.jp/). “Ratio” was calculated by dividing the number of cis-elements in gene of rice by the number of cis-elements in gene
of Arabidopsis. ND ⫽ not determined. Cis-element function was classified (Category) on the basis of the annotation of the element entry in the PLACE database.

common elements that were present in at least one gene in both exist in rice. However, the number of cis-elements for
species. protein storage was remarkably rich in both species (“com-
Six kinds of cis-elements for dehydration-stress re- mon” in “specific or common” column in Table 5), and the
sponse (ACGTATERD1, MYB1AT, ABRELATERD1, number of other elements for protein storage (⫺300ELE-
MYB2CONSENSUSAT, MYCATERD1, MYCCONSEN- MENT, RYREPEATGMGY2, RYREPEATLEGUMIN-
SUSAT) were specified as elements in Arabidopsis (italic BOX, RYREPEATBNNAPA) were richer in the upstream
characters in “cis-element” column in Table 5). The speci- regions of genes of rice than in those of Arabidopsis. We
ficities of Arabidopsis cis-elements for dehydration stress suggest that Arabidopsis might use these conserved ele-
might be derived from differences in the growth environ- ments for protein storage. The specificities of rice in protein
ments of each species. These six kinds of elements do not storage might be derived so that structure of rice seed is
Physiol Genomics • VOL 17 • www.physiolgenomics.org
98 PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE

Table 6. Comparison of cDNA array, 60-mer oligo array and Q-RT-PCR relative expression results
for ABA responsive transcripts
Q-RT-
cDNA Array 60-mer Oligo Array PCR

Category Putative gene ID Clone Name Accession No. Ratio Oligo Array Ratio Ratio

Cell growth Aldose-reductase-related protein (Avena fatua) CH2836 AU101007 4.47 J013074I15 8.21 9.99
Cell growth Glucose dehydrogenase (Hordeum vulgare) EC0269 C19333 4.56 002-169-E03 9.39 19.40
Cell growth Lipid transfer protein (Oryza sativa) CH3458 AU063655 2.17 J033094A19 1.03
Cell growth Lipid transfer protein (O. sativa) ST3943 D41439 2.17 006-205-A04 0.93 6.12
Cell growth Mitochondrial phosphate transporter (O. sativa) EC0376 C99183 2.84 J023022H13 0.84 4.9
Cell growth myo-inositol phosphate synthase, RINO1 (O. sativa) CH0155 C97424 2.41 001-019-H08 2.08 7.77
Cell growth myo-inositol phosphate synthase, RINO1 (O. sativa) CB2444 AU068211 2.37 001-019-H08 2.08 1.00
Cell growth myo-inositol phosphate synthase, RINO1 (O. sativa) FE0401 AU174807 2.16 001-019-H08 2.08 5.86
Cell growth Proteinase inhibitor, RPI (O. sativa) EC1619 AU172733 7.14 002-125-A10 1.02 93.11
Defense 16 kDa oleosin, ole16 (O. sativa) EC0540 C19519 3.01 J03317E10 1.60
Defense 16 kDa oleosin, ole16 (O. sativa) EC1755 C20257 2.45 J033127E10 1.60 18.02
Defense Hydrophobic LEA-like protein (O. sativa) CH2085 C28696 3.64 001-118-B01 4.76 5.71

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Defense LEA group 3 protein (O. sativa) EA0201 AU174470 4.53 001-124-D08 1.70 6.86
Defense Probenazole-inducible protein, PBZ1 (O. sativa) SA1317 AU057293 2.06 J023104B18 4.44 16.30
Defense Water-stress-regulated gene (O. sativa) EA0326 AU174486 3.01 001-125-H02 1.44 4.81
Germination Embryo globulin 1 (Hordeum vulgare) CH2882 AU063570 5.95 001-119-A01 5.07 44.51
Germination Osem gene (O. sativa) EC1638 C20180 5.77 001-119-E02 2.35 6.27
Maintenance Thionin (O. sativa) ST1215 D39662 2.12 001-107-G07 1.09 2.88
Maintenance Thionin (O. sativa) ST3801 D41338 1.98 001-107-G07 1.09 4.18
Transport Abscisic acid and stress inducible gene (H. vulgare) EB1768 AU069752 2.05 001-125-D01 4.11
Unclassified Novel protein, osr40c1 (O. sativa) EE0241 AU165969 2.19 J023031K10 1.70 1.89
Unclassified RAB24 (O. sativa) CH1659 C28576 5.16 J033115O22 4.46 6.92
Unclassified Unknown CH0161 AU091293 2.21 001-207-H07 1.62 1.00
Unclassified Unknown CH0445 C28247 3.41 001-117-H11 3.80 15.42
Unclassified Unknown CH1555 AU166971 3.80 J033118I15 0.89 33.49
Unclassified Unknown CH3201 C29020 3.06 001-117-H11 3.80 387.24
Unclassified Unknown CH3541 C29099 2.08 001-122-F11 0.79
Unclassified Unknown EC0136 C19228 5.66 J033025I17 0.88 1.00
Unclassified Unknown EC0512 AU172707 5.52 001-118-F01 0.91 7.83
Unclassified Unknown EC0769 C19677 5.35 J023009K15 0.74 1.79
Unclassified Unknown EC0946 C19796 7.12 001-118-E12 7.59 48.18
Unclassified Unknown EC1146 C19868 3.66 001-119-E08 2.18 25.1
Unclassified Unknown EC1152 C19872 2.40 002-133-B10 4.13 2.29
Unclassified Unknown EC1222 C19919 2.41 002-132-C06 1.03
Unclassified Unknown EC1379 C20014 2.22 002-102-E03 0.99 1.98
Unclassified Unknown EC1470 C20073 2.73 002-135-G05 3.91 14.43
Unclassified Unknown EC1656 C20195 2.58 001-120-D12 15.62 25.15
Unclassified Unknown EC1996 C20419 3.11 002-137-B01 3.47 11.93
Unclassified Unknown ED1001 C99650 2.44 J023019B19 2.50 0.00
Unclassified Unknown EE1762 AU029786 3.82 001-038-G02 1.03 1.00
cDNA microarray data for a set of transcripts previously reported as ABA responsive, modified here from Yazaki et al. (38), were compared to rice oligo
microarray and quantitative real-time PCR (Q-RT-PCR) expression data. Only those 40 transcripts from the original list that are represented on the 8,987 cDNA
microarray, and on the 60-mer oligo microarray, are shown. We selected 40 transcripts with significant expression differences of median ⫾ 2SD over a normal
distribution in cDNA array analysis. We amplified Q-RT-PCR measurements of 35 transcripts using specific primers that designated from EST clones on cDNA
microarray. Expression ratios are expressed as ABA treatment callus vs. nontreatment callus, and nonsignificant results are italicized.

different from that of Arabidopsis. Also, that the cis-element all types of cis-elements of GA-responsive genes in rice and
for expression of the amylase gene (CGACGOSAMY3) was Arabidopsis are shown in Supplemental Tables S8 (rice) and
specified as an element in rice may suggest the view that the S9 (Arabidopsis). The results of comparison between 151
difference in these elements is derived from differences in clones responsive to GA in rice and 117 clones of corre-
organization between rice and Arabidopsis. Although the sponding Arabidopsis genes are summarized in Supplemen-
cis-element MYBCORE for water-stress response appears in tal Table S10. The comparison of cis-elements for GA-
both species as a common cis-element in Table 5, the responsive genes between rice and Arabidopsis gives a
number of elements in rice was 1.34 times that in Arabi- result similar to that for ABA. These differences in cis-
dopsis. These results suggest that rice might use a slightly elements for protein storage and dehydration-stress response
different mechanism for response to water stress. Two kinds between rice and Arabidopsis may have accumulated
of ABA-responsive element (RAV1AAT, DPBFCORED- through differences in the organization of each plant or
CDC3) and one GA-responsive element (DOFCOREZM) through evolutionary responses to the growth environment.
were rich in cis-elements conserved between rice and Ara- The comparative analysis of cis-elements among various
bidopsis (“common” in “specific or common” column in species for the detection of characteristic in plants may
Table 5). We suggest that each species might have a com- become a useful indicator for the more efficient creation
mon pathway for ABA or GA metabolism. The numbers of of transgenic plants. The comparison also may support the
Physiol Genomics • VOL 17 • www.physiolgenomics.org
PROFILING OF PHYTOHORMONE-RESPONSIVE GENES IN RICE 99
prediction of the physiological functions of the products ABA and GA response in rice. We identified genes that had
of “unknown” genes. In promoter sequences search of never before been annotated as ABA or GA responsive in other
the ABA response genes obtained, we detected that several reports of comprehensive expression analysis of ABA-respon-
rice genes (AK064966, AK073100, AK073380, AK073777, sive genes in plant (12, 29, 33), detected new interactions
AK073833, AK102307, AK103170, AK105316, AK106508, between genes responsive to the two hormones, comprehen-
AK108159, AK110259, and AK110912 in Supplemental Table sively characterized cis-elements of hormone-responsive
S6) did not contain any ABA-responsive element in their genes, obtained new putative gene functions from phenotypic
promoters on rice genome as well as Arabidopsis report (33). results, and characterized cis-elements of rice and Arabidopsis.
These results suggest the existence of novel cis-acting elements The results revealed that our tools and methods of functional
involved in ABA-inducible gene expression in their promoters genomics can identify genes that control particular phenotypes
of rice and Arabidopsis. In our study, WRKY transcription and can identify the transcriptional regulator (cis-element) of
factor (AK073100, AK110912 in Supplemental Table S6), a rice faster and more accurately than ever before. For the most
protein known to mediate the pathogen-induced defense pro- effective functional analysis of the genome, all available in-
gram, were induced by ABA treatment in rice callus. The data formation needs to be integrated. Systematically connecting
suggest that these not only reveal the cross talk between ABA powerful tools and information for functional genomics of rice
response pathway and pathogen-induced defense program but will allow researchers in the life sciences, especially crop

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also support the suggestion that W-box binding motif se- science, to change the direction of research. All of the infor-
quences, most of the WRKY proteins showed to bind to the mation on rice functional genomics used in this report can be
sequence, are a novel cis-acting element involved in ABA- accessed through the Rice PIPELINE (http://cdna01.
inducible gene expression (33). dna.affrc.go.jp/PIPE/) (40), including the databases KOME,
PLACE, Tos17, Integrated Rice Genome Explorer (31) (INE:
Comparison of ABA-Responsive Genes Among 60-mer http://rgp.dna.affrc.go.jp/giot/INE.html), and the Rice Ex-
Oligonucleotide Array, cDNA Array, and Quantitative pression Database (RED; http://red.dna.affrc.go.jp/RED/)
RT-PCR (39). Details of all of our materials, including FL-cDNA and
We previously identified a set of transcripts that were more mutant lines, can be obtained from the Rice Genome Resource
abundant in ABA-treated callus than in untreated callus by Center (http://www.rgrc.dna.affrc.go.jp).
using rice 8,987-cDNA microarray (38). To assess the utility of ACKNOWLEDGEMENTS
our oligonucleotide array in a practical context (i.e., are the
We thank Dr. Hisako Ooka, Dr. Toshifumi Nagata, Dr. Hitomi Yamada,
same genes identified?), we compared expression ratios deter- and Masaki Shimono (NIAS) for helpful comments. We also thank Kanako
mined by cDNA microarray and oligonucleotide array for Shimbo, Yumiko Yoshida (Institute of the Society for Techno-innovation of
ABA-responsive transcripts (Table 6). Agriculture, Forestry and Fisheries), Dr. Naoki Kishimoto, Sachiko Honda,
The sequences of EST clones on rice 8,987-cDNA microar- Ayano Endo, Yuki Sato, Chikako Miyamoto, Kazuko Toyoshima, and Keiko
ray were searched for in the rice FL-cDNA database, KOME Takeuchi (NIAS) for helpful support. We also thank Chao Jie Li and Makoto
Yamamoto (Hitachi Software Engineering Co. Ltd.) for technical support.
(http://cdna01.dna.affrc.go.jp/cDNA/), which includes a col-
lection of about 32,000 unique FL-cDNAs. We obtained 36 GRANTS
FL-cDNAs linking with the oligonucleotide array correspond- This work was supported by a grant from the Ministry of Agriculture,
ing to the 40 EST clones selected as responsive to ABA Forestry, and Fisheries of Japan (Rice Genome Project MA-1000).
treatment in the cDNA microarray analysis (38) by BLAST
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