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The Plant Cell, Vol. 13, 61–72, January 2001, www.plantcell.org © 2001 American Society of Plant Physiologists
Monitoring the Expression Pattern of 1300 Arabidopsis Genes
under Drought and Cold Stresses by Using a Full-Length
cDNA Microarray
Motoaki Seki,
a,b
Mari Narusaka,
a
Hiroshi Abe,
c
Mie Kasuga,
c
Kazuko Yamaguchi-Shinozaki,
c
Piero Carninci,
d
Yoshihide Hayashizaki,
d
and Kazuo Shinozaki
a,b,1
a
Plant Mutation Exploration Team, Plant Functional Genomics Research Group, RIKEN Genomic Sciences Center, 3-1-1
Koyadai, Tsukuba 305-0074, Japan
b
Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba 305-0074, Japan
c
Biological Resources Division, Japan International Research Center for Agricultural Sciences, Ministry of Agriculture,
Forestry, and Fisheries, 2-1 Ohwashi, Tsukuba, Ibaraki 305-0851, Japan
d
Genome Science Laboratory, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba 305
-
0074, Japan
Full-length cDNAs are essential for functional analysis of plant genes. Using the biotinylated CAP trapper method, we
constructed full-length Arabidopsis cDNA libraries from plants in different conditions, such as drought-treated, cold-
treated, or unstressed plants, and at various developmental stages from germination to mature seed. We prepared a
cDNA microarray using
ⵑ
1300 full-length Arabidopsis cDNAs to identify drought- and cold-inducible genes and target
genes of DREB1A/CBF3, a transcription factor that controls stress-inducible gene expression. In total, 44 and 19 cDNAs
for drought- and cold-inducible genes, respectively, were isolated, 30 and 10 of which were novel stress-inducible genes
that have not been reported as drought- or cold-inducible genes previously. Twelve stress-inducible genes were identi-
fied as target stress-inducible genes of DREB1A, and six of them were novel. On the basis of RNA gel blot and microar-
ray analyses, the six genes were identified as novel drought- and cold-inducible genes that are controlled by DREB1A.
Eleven DREB1A target genes whose genomic sequences have been registered in the GenBank database contained the
dehydration-responsive element (DRE) or DRE-related CCGAC core motif in their promoter regions. These results show
that our full-length cDNA microarray is a useful material with which to analyze the expression pattern of Arabidopsis
genes under drought and cold stresses, to identify target genes of stress-related transcription factors, and to identify
potential
cis
-acting DNA elements by combining the expression data with the genomic sequence data.
INTRODUCTION
Sequencing projects are producing large quantities of ge-
nomic and cDNA sequences for a number of organisms. In
the model plant Arabidopsis, the complete genomic se-
quences of two chromosomes have been determined (Lin et
al., 1999; Mayer et al., 1999), and the entire genomic se-
quence was completed by the end of 2000. Expressed se-
quence tag (EST) projects have also provided a major
contribution with the discovery of expressed genes (Höfte et
al., 1993; Newman et al., 1994; Cooke et al., 1996; Asamizu
et al., 2000). A recent release of dbEST (the EST database of
the National Center for Biotechnology Information, http://
www.ncbi.nlm.nih.gov) contained partial cDNA sequences
in which more than half of the total gene complement (i.e.,
ⵑ
28,000 genes) is represented (as estimated from the gene
content of the entirely sequenced chromosome 2 in Arabi-
dopsis [Lin et al., 1999]). A major challenge for the coming
decade is the functional analysis of this large set of genes.
Recently, microarray technology has become a useful tool
for the analysis of genome-scale gene expression (Schena
et al., 1995; Eisen and Brown, 1999). This DNA chip-based
technology arrays cDNA sequences on a glass slide at a
density
⬎
1000 genes/cm
2
. These arrayed sequences are
hybridized simultaneously to a two-color fluorescently la-
beled cDNA probe pair prepared from RNA samples of dif-
ferent cell or tissue types, allowing direct and large-scale
comparative analysis of gene expression. This technology
was first demonstrated by analyzing 48 Arabidopsis genes
for differential expression in roots and shoots (Schena et al.,
1995). Microarrays were used to study 1000 randomly cho-
sen clones from a human cDNA library for identification of
1
To whom correspondence should be addressed. E-mail shinozaki@
rtc.riken.go.jp; fax 81-298-36-9060.
62 The Plant Cell
novel genes responding to heat shock and protein kinase C
activation (Schena et al., 1996). In another study, expression
profiles of inflammatory disease-related genes were ana-
lyzed under various induction conditions by this chip-based
method (Heller et al., 1997). Furthermore, the yeast genome
of
⬎
6000 coding sequences has been analyzed for dynamic
expression by the use of microarrays (DeRisi et al., 1997;
Wodicka et al., 1997). However, in plant science, only sev-
eral reports of microarray analyses have been published
(Schena et al., 1995; Ruan et al., 1998; Aharoni et al., 2000;
Reymond et al., 2000). We constructed Arabidopsis full-
length cDNA libraries (Seki et al., 1998) from plants in differ-
ent conditions, such as drought-treated and cold-treated
plants, by using the biotinylated CAP trapper method
(Carninci et al., 1996). In this study, we prepared an Arabi-
dopsis full-length cDNA microarray using
ⵑ
1300 full-length
cDNAs, including stress-inducible genes, to monitor the ex-
pression patterns of genes under drought and cold stresses.
Plant growth is greatly affected by environmental abiotic
stresses, such as drought, high salinity, and low tempera-
ture. Plants respond and adapt to these stresses to survive
under stress conditions. Among these abiotic stresses,
drought or water deficit is the most severe limiting factor of
plant growth and crop production. Drought stress induces
various biochemical and physiological responses in plants.
Recently, a number of genes have been described that re-
spond to drought at the transcriptional level (Bohnert et al.,
1995; Ingram and Bartels, 1996; Bray, 1997; Shinozaki and
Yamaguchi-Shinozaki, 1997, 1999, 2000). Stress-inducible
genes have been used to improve the stress tolerance of
plants by gene transfer (Holmberg and Bülow, 1998; Bajaj et
al., 1999). It is important to analyze the functions of stress-
inducible genes not only to understand the molecular mech-
anisms of stress tolerance and the responses of higher
plants but also to improve the stress tolerance of crops by
gene manipulation. Hundreds of genes are thought to be in-
volved in abiotic stress responses. In the present study, we
applied cDNA microarray analysis to identify new drought-
or cold-inducible genes.
Dehydration-responsive element/C-repeat (DRE/CRT) has
been identified as an important
cis
-acting element in
drought-, high salt–, and cold stress–responsive gene
expression in an abscisic acid (ABA)–independent manner
(Yamaguchi-Shinozaki and Shinozaki, 1994; Thomashow,
1999; Shinozaki and Yamaguchi-Shinozaki, 2000). Tran-
scription factors (DREB/CBF) involved in DRE/CRT-respon-
sive gene expression have been cloned (Stockinger et al.,
1997; Gilmour et al., 1998; Liu et al., 1998; Shinwari et al.,
1998). DREB1/CBFs are thought to function in cold-respon-
sive gene expression, whereas DREB2s are involved in
drought-responsive gene expression. Strong tolerance to
freezing stress was observed in transgenic Arabidopsis
plants that overexpress
CBF1
(
DREB1B
) cDNA under the
control of the cauliflower mosaic virus (CaMV) 35S promoter
(Jaglo-Ottosen et al., 1998). We reported that overexpres-
sion of the
DREB1A
(
CBF3
) cDNA under the control of the
CaMV 35S promoter or the stress-inducible
rd29A
promoter
in transgenic plants gave rise to strong constitutive expres-
sion of the stress-inducible DREB1A target genes and in-
creased tolerance to freezing, drought, and salt stresses (Liu
et al., 1998; Kasuga et al., 1999). Previously (Kasuga et al.,
1999), we identified six DREB1A target genes:
rd29A/lti78/
cor78
,
kin1
,
kin2/cor6.6
,
cor15a
,
rd17/cor47
, and
erd10
.
However, it is not well understood how overexpression of
the
DREB1A
cDNA in transgenic plants increases stress tol-
erance to freezing, drought, and salt. To study the molecular
mechanisms of drought and freezing tolerance, it is impor-
tant to identify and analyze more genes that are controlled
by DREB1A. Therefore, in the present study, we used cDNA
microarray analysis to identify novel DREB1A target genes.
A complete list of
ⵑ
1300 Arabidopsis cDNA clones and the
expression data from this study are available at http://rtcweb.
rtc.riken.go.jp/lab/pmb.
RESULTS
Arabidopsis Full-Length cDNA Microarray
Using the biotinylated CAP trapper method, we constructed
full-length cDNA libraries from Arabidopsis plants in differ-
ent conditions, such as drought-treated, cold-treated, and
unstressed plants, at various developmental stages from
germination to mature seed (Seki et al., 1998). From the full-
length cDNA libraries, we isolated
ⵑ
1300 independent Ara-
bidopsis full-length cDNAs. We used a method described
previously (Eisen and Brown, 1999) to array polymerase
chain reaction (PCR)–amplified cDNA fragments onto glass
slides. We prepared a full-length cDNA microarray contain-
ing 1300 Arabidopsis full-length cDNAs, including drought-
inducible genes
responsive to dehydration
(
rd
) and
early re-
sponsive to dehydration
(
erd
) (Taji et al., 1999), as positive
controls, the Arabidopsis
␣
-tubulin gene (Ludwig et al.,
1987) with same expression level in our experimental condi-
tions as an internal control, and the mouse nicotinic acetyl-
choline receptor epsilon-subunit (nAChRE) gene and the
mouse glucocorticoid receptor homolog gene, which have
no substantial homology to any sequences in the Arabi-
dopsis database, to assess for nonspecific hybridization
as negative controls. To assess the reproducibility of the
microarray technique, we arrayed the PCR products from
each cDNA clone twice on each slide.
Isolation of Drought- and Cold-Inducible Genes by Use
of the cDNA Microarray
The cDNA microarray hybridized with Cy3 and Cy5 fluores-
cently labeled probe pairs of drought-treated plants plus
unstressed plants and cold-treated plants plus unstressed
Arabidopsis Full-Length cDNA Microarray 63
plants prepared as described in Methods. Figure 1 shows
an image of a portion of the cDNA microarray. Dual label-
ing of cDNA probe pairs with Cy3-dUTP for one mRNA
sample and Cy5-dUTP for the other sample allows simul-
taneous hybridization to DNA elements on microarrays and
facilitates direct quantitative measurements of gene expres-
sion between two different conditions, stressed and un-
stressed. Hybridized microarrays were scanned by two
separate laser channels for Cy3 and Cy5 emissions from
each DNA element. The ratio of the two fluorescent signal
intensities of each DNA element was then measured as a
relative measure to determine changes in the differential
expression of genes represented by cDNA spots on the mi-
croarrays. In this study, we used the
␣
-tubulin gene as an
internal control gene because its expression level is al-
most the same in the two experimental conditions we ana-
lyzed.
Figure 2 shows the strategy for identification of drought-
or cold-inducible genes. mRNAs from drought- or cold-
treated plants and wild-type unstressed plants were used
for the preparation of Cy3-labeled and Cy5-labeled cDNA
probes, respectively. These cDNA probes were mixed and
hybridized with the cDNA microarray. To assess the repro-
ducibility of the microarray analysis, we repeated the same
experiment five times (see Table 2). Hybridization of differ-
ent microarrays with the same mRNA samples indicated
good correlation. We regarded genes with an expression ra-
tio (drought/unstressed or cold/unstressed) greater than
twofold that of the
␣
-tubulin gene as drought- or cold-
inducible genes.
Drought- and Cold-Inducible Genes Identified with the
Full-Length cDNA Microarray
In total, 44 drought-inducible genes were identified by cDNA
microarray analysis (Tables 1 and 2). Fourteen of these
genes were reported to be drought-inducible genes, such
as
rd29A/cor78
,
cor15a
,
kin1
,
kin2
,
rd17/cor47
,
erd10
, and
rd20
(Kiyosue et al., 1994a; Bohnert et al., 1995; Ingram and
Bartels, 1996; Bray, 1997; Shinozaki and Yamaguchi-
Shinozaki, 1997, 1999, 2000; Taji et al., 1999; Takahashi et
al., 2000). These results showed that our cDNA microarray
system functioned properly to find stress-inducible genes.
Among the 30 new drought-inducible genes that have not
been reported previously as drought inducible, we found
cDNAs (FL3-5A3, FL6-55, FL5-1N11, FL5-2O24, FL5-2H15,
and FL1-159) that are identical at the nucleotide level with pu-
tative cold acclimation protein (accession number AC006438),
LEA 76 type 1 protein (accession number X91919), nonspe-
cific lipid transfer protein (LTP1; accession number M80567),
putative water channel protein (accession number AC005770),
T45998 EST, and HVA22 homolog (accession number
AB015098).
Also, 19 cold-inducible genes were identified by the cDNA
microarray analysis (Tables 1 and 2). Among them, nine
were reported to be cold-inducible genes:
rd29A
,
cor15a
,
kin1
,
kin2
,
rd17
,
erd10
,
erd7
,
erd4
(Kiyosue et al., 1994b;
Shinozaki and Yamaguchi-Shinozaki, 1997, 1999, 2000; Taji
et al., 1999; Thomashow, 1999), and
DREB1A
(Liu et al.,
1998). Among the 10 new cold-inducible genes that have
not been reported as cold inducible previously, we found
cDNAs (FL3-5A3, FL5-3A15, FL5-3P12, FL5-90, FL5-2I22,
and FL1-159) that are identical with putative cold acclimation
protein (accession number AC006438), ferritin (accession
number X94248), EXGT-A2 (accession number D63510),

-amylase (accession number AJ250341), DC 1.2 homolog
(accession number X80342), and HVA22 homolog (acces-
sion number AB015098) and cDNAs (FL5-1A9, FL5-95, and
FL5-3M24) showing sequence similarity with nodulin-like
protein (accession number CAA22576), rice glyoxalase I (ac-
cession number AB017042), and LEA protein homolog (SAG21;
accession number AF053065).
Stress-Inducible Genes That Are Controlled by the
DREB1A Transcription Factor
We used the full-length cDNA microarray to identify stress-
inducible genes that are controlled by the DREB1A tran-
scription factor. The strategy for identification of DREB1A
target genes is shown in Figure 2. mRNAs prepared from
transgenic Arabidopsis plants that overexpress the
DREB1A
Figure 1. cDNA Microarray Analysis of Gene Expression under Cold
Stress.
A fluorescently labeled cDNA was prepared from mRNA isolated
from unstressed Arabidopsis plants by reverse transcription in the
presence of Cy5-dUTP. A second probe, labeled with Cy3-dUTP,
was prepared from cold-treated (2 hr) plants. After simultaneous hy-
bridization of both probes with a cDNA microarray containing ⵑ1300
Arabidopsis cDNA clones, a pseudocolor image was generated.
Genes induced and genes repressed after cold stress are repre-
sented as red and green signals, respectively. Genes expressed at
approximately equal levels between treatments appear as yellow
signals. The intensity of each spot corresponds to the absolute
amount of expression of each gene. Cold-inducible genes (rd29A)
are shown as red signals. ␣-Tubulin genes (internal control) are
shown as yellow signals. No signals are shown for nAChRE. Bar ⫽
300 m.
64 The Plant Cell
cDNA under the control of the CaMV 35S promoter
(35S:DREB1A transgenic plants) and wild-type control
plants were used for the preparation of Cy3-labeled and
Cy5-labeled cDNA probes, respectively. These cDNA probes
were mixed and hybridized with the cDNA microarray. We
regarded genes with expression levels more than two times
greater in the 35S:DREB1A transgenic plants than in wild-
type control plants as DREB1A target genes.
In total, 12 DREB1A target genes were identified by cDNA
microarray analysis (Tables 1 and 2). Among them, six were
reported to be DREB1A target genes:
rd29A/cor78
,
cor15a
,
kin1
,
kin2
,
rd17/cor47
, and
erd10
(Kasuga et al., 1999). Also,
among the six novel DREB1A target genes that have not
been reported as DREB1A target genes previously, we found
cDNAs (FL3-5A3, FL5-2I22, FL5-94, and FL5-77) showing
sequence identity with putative cold acclimation protein (ac-
cession number AC006438), DC 1.2 homolog (accession
number X80342), enolase (accession number X58107), and
peroxiredoxin TPX1 (accession number AF121355) and a
cDNA (FL3-27) showing sequence similarity with cowpea
cysteine proteinase inhibitor (accession number Z21954)
and
erd4
cDNA (Kiyosue et al., 1994b; Taji et al., 1999). The
relationship between the six novel DREB1A target genes
and stress tolerance is unelucidated and should be ana-
lyzed in the future.
RNA Gel Blot Analysis
In the cDNA microarray analysis, it is important to extract
correct data with minimal effort. We evaluated the validity of
the cDNA microarray analysis by the following methods. Ini-
tially, we identified 80 genes whose expression ratios (dehy-
dration for 2 hr/unstressed) were more than twice that of
␣
-tubulin. We performed RNA gel blot analysis of 80 puta-
tive drought-inducible genes and identified 44 as real
drought-inducible genes. The inconsistency between the mi-
croarray results and the RNA gel blot results on several
genes was due to (1) weak expression of genes, (2) high
background, (3) dust or scratches on the cDNA spots, and
(4) bad cDNA probes with low specific activity. Therefore,
we flagged the experimental data mentioned above and ex-
cluded half of them from the subsequent analysis. After this
data treatment, we identified 44 drought-inducible genes,
19 cold-inducible genes, and 12 DREB1A target genes
based on cDNA microarray analysis. Then we performed
RNA gel blot analysis to confirm the results obtained with
the cDNA microarray. The results of expression data ob-
tained by microarray analysis were in agreement with those
obtained by RNA gel blot analysis in 44 drought-inducible
genes, 19 cold-inducible genes, and 12 DREB1A target
genes identified (data not shown). In Figure 3, the results of
the microarray analysis are compared with those of RNA gel
blot analysis for the six new DREB1A target genes (FL3-5A3,
FL3-27, FL5-2I22, FL5-94, FL5-77, and
erd4
). All six
genes were induced by dehydration and cold treatment
Figure 2. Strategy for the Identification of Drought- or Cold-Induc-
ible Genes and DREB1A Target Genes.
(1) mRNAs from dehydrated or cold-treated plants and wild-type
(WT) unstressed plants were used for preparation of Cy3-labeled
and Cy5-labeled cDNA probes, respectively. These cDNA probes
were mixed and hybridized with the cDNA microarray. In this study,
we used the ␣-tubulin gene as an internal control gene because its
expression level is almost the same in the two conditions. We re-
garded the genes with expression ratios (drought/unstressed o
r
cold/unstressed) greater than twice that of ␣-tubulin as drought- or
cold-inducible genes.
(2) mRNAs from 35S:DREB1A transgenic plants and wild-type (WT)
unstressed plants were used for preparation of Cy3-labeled and
Cy5-labeled cDNA probes, respectively. These cDNA probes were
mixed and hybridized with the cDNA microarray. In this study, we
used the ␣-tubulin gene as an internal control gene because its ex-
pression level is almost the same in the two conditions. We regarded
the genes with expression levels more than two times greater in
35S:DREB1A transgenic plants than in wild-type unstressed plants
as DREB1A target genes.
Arabidopsis Full-Length cDNA Microarray 65
and overexpressed in the 35S:DREB1A plants under un-
stressed conditions.
DISCUSSION
Many Novel Drought- or Cold-Inducible Genes Could Be
Identified with the Full-Length cDNA Microarray
Hundreds of plant genes are thought to be induced by
stress, such as drought, high salinity, and low temperature,
and function in the stress tolerance and responses of plants.
However, only
ⵑ
50 drought- or cold-inducible genes have
been reported to date (Shinozaki and Yamaguchi-Shinozaki,
1999; Thomashow, 1999). On the basis of the microarray
and RNA gel blot analyses (drought 2 hr, 10 hr and cold 2 hr,
10 hr) of the drought- or cold-inducible genes identified,
they were grouped as follows: (1) drought- and cold-induc-
ible genes, (2) drought-specific inducible genes, and (3)
cold-specific inducible genes. As a result, 20 of them are
drought- and cold-inducible genes, five of them are
drought-specific inducible genes, including four novel genes
(FL6-55, FL5-2D23, FL2-56, and FL5-3J4), and two of them
are cold-specific inducible genes, including one novel gene
(FL5-90). However, 21 of the drought- or cold-inducible
genes were difficult to classify into the three groups because
of weak expression of genes or high background (Table 2
and Figure 4). On the basis of a search for homology in the
GenBank database using the BLAST program, two cDNAs
(FL6-55 and FL2-56) for drought-specific inducible genes
were found to have sequence identity with LEA 76 type I pro-
tein (accession number X91919) and glycine-rich protein 3
short isoform (GRP3S; accession number AF104330); two
drought-specific cDNAs (FL5-3J4 and FL5-2D23) were found
to have sequence similarity to
Borrelia burgdorferi
heat
shock protein dnaJ (accession number M96847) and T20517
EST; and one cDNA (FL5-90) for cold-specific inducible genes
was found to have sequence identity with the gene for

-amy-
lase (accession number AJ250341). Analyses of these novel
drought- or cold-specific inducible genes will provide infor-
mation about genes involved in stress tolerance and
cis
-
acting promoter elements that function in drought- or cold-
specific gene expression.
Most of the identified genes are induced by both drought
and cold stress (Table 2 and Figure 4) and contain the DRE
or DRE-related CCGAC core motif in their promoters, as de-
scribed below. One of the drought-specific inducible genes
is
rd20
, which encodes a Ca
2
⫹
binding protein. The
rd20
gene is induced by dehydration and high salinity but not by
cold stress (Takahashi et al., 2000). The
rd20
gene is in-
duced by ABA treatment and contains the ABA-responsive
element (ABRE) motif in its promoter, which indicates that
part of drought-specific gene expression is regulated by
an ABA-dependent pathway (Shinozaki and Yamaguchi-
Shinozaki, 2000). In contrast, one of the cold-specific induc-
ible genes encodes DREB1A/CBF3, a cold-inducible tran-
scription factor with the AP2/ERF domain. The
DREB1A/
CBF3
promoter is thought to contain
cis
-acting elements in-
volved in cold-specific gene expression (Gilmour et al.,
1998; Shinwari et al., 1998). These results support the exist-
ence of different regulatory systems in drought- and cold-
inducible gene expression (Shinozaki and Yamaguchi-
Shinozaki, 2000).
Most of the Drought- and Cold-Inducible Genes Are
Target Genes of DREB1A/CBF3
We have reported the presence of at least four signal trans-
duction pathways that function in the activation of stress-
inducible genes under drought conditions, two of which are
ABA dependent and two of which are ABA independent
(Shinozaki and Yamaguchi-Shinozaki, 1997, 1999, 2000). To
understand the molecular mechanisms of gene expres-
sion in response to water stress, studies of
cis-
and
trans-
acting elements are important. A conserved sequence,
PyACGTG(G/T)C, has been reported to function as an ABRE
in many ABA-responsive genes (Ingram and Bartels, 1996;
Bray, 1997; Shinozaki and Yamaguchi-Shinozaki, 1999).
Also, a 9-bp conserved sequence, TACCGACAT, named
the dehydration-responsive element (DRE), has been re-
ported to be essential for regulation of the induction of
rd29A
expression under drought, low temperature, and high
salt stress conditions, but it does not function as an ABRE
(Yamaguchi-Shinozaki and Shinozaki, 1994). DRE and
DRE-related core motifs (CCGAC), CRT, and LTRE have
also been reported in the promoter regions of drought- and
cold-inducible genes such as
kin1
,
kin2
,
rd17/cor47
, and
cor15a
(Table 3) (Baker et al., 1994; Wang et al., 1995;
Iwasaki et al., 1997).
In this study, we identified 12 DREB1A target genes by
cDNA microarray analysis. Among the six new DREB1A target
Table 1.
Drought- and Cold-Inducible Genes, and DREB1A Target
Genes Identified by cDNA Microarray Analysis
Genes No. of Genes
Drought-inducible genes 44
New drought-inducible genes
a
30
Cold-inducible genes 19
New cold-inducible genes
b
10
DREB1A target genes 12
New DREB1A target genes
c
6
a
Drought-inducible genes that have not been reported as drought-
inducible genes previously.
b
Cold-inducible genes that have not been reported as cold-induc-
ible genes previously.
c
DREB1A target genes that have not been reported as DREB1A tar-
get genes previously.
66 The Plant Cell
Arabidopsis Full-Length cDNA Microarray 67
genes, genomic sequences of the five target genes were
registered in the GenBank database in October 2000. A 9-bp
DRE sequence was observed in the promoter regions of genes
corresponding to FL3-5A3 and FL3-27 cDNAs (Table 3). The
CCGAC core sequence was observed in the promoter regions
of genes corresponding to FL3-5A3, FL5-2I22, FL5-77, and
FL5-94 cDNAs (Table 3). Most of the drought- and cold-
inducible genes are DREB1A target genes and contain DRE
or DRE-related CCGAC core motifs in their promoters (Table
3 and Figure 4). The ABRE sequence, PyACGTG(G/T)C, was
observed in the promoter regions of six of the 12 DREB1A
target genes identified (Table 3). This finding indicates that
many drought- and cold-inducible genes are controlled by
both ABA-dependent and ABA-independent pathways.
However, expression of several drought- and cold-inducible
genes (FL5-3M24, FL5-3A15, FL5-1A9, and FL5-2O24) was
not increased in the 35S:DREB1A transgenic plants (Figure
4), indicating that these genes are not DREB1A target
genes. The CCGAC core sequence was not observed in the
2000-bp upstream region of the 5
⬘
terminus of the FL5-
3M24 cDNA. These results suggest the existence of novel
cis
-acting elements involved in drought- and cold-inducible
gene expression. By comparing the Arabidopsis full-length
cDNA sequences with the Arabidopsis genomic sequences
(the entire Arabidopsis genomic sequence was completed
by the end of 2000), the promoter sequences and
cis
-acting
elements of each gene can be studied on the basis of full-
length cDNA sequences. These analyses will provide more
information on gene expression and signal transduction in
abiotic stress responses.
In the present study, we identified 12 DREB1A target
genes containing six new target genes by using cDNA mi-
croarray analysis. However, the cDNA microarray analysis
does not permit us to distinguish direct targets from indirect
targets of the
DREB1A
gene. Resolving this issue and the
complex interrelationships among the induced genes will re-
quire another approach, such as analysis for cis-acting ele-
ments or gel shift assays by using promoter regions of each
stress-inducible gene.
Advantages and Disadvantages of the Full-Length
cDNA Microarray
Several reports have been published on the use of plant
EST microarrays. In the present study, we used an Arabi-
dopsis full-length cDNA microarray, which has several ad-
vantages. Using the full-length cDNA microarray, it is easy
to isolate full-length cDNAs for further functional analysis.
Also, by comparing the Arabidopsis full-length cDNA se-
quences with the Arabidopsis genomic sequences, the pro-
moter sequences and cis-acting elements of each gene can
be studied on the basis of full-length cDNA sequences.
Moreover, there is little cross-hybridization with pseudo-
genes compared with using the genomic DNA array. How-
ever, cross-hybridization between highly related sequences
may occur in the full-length cDNA microarray (Richmond
and Somerville, 2000). To avoid cross-hybridization prob-
lems, the use of oligonucleotide microarrays such as the Af-
fymetrix GeneChip Array (Affymetrix, Inc., Santa Clara, CA)
may be appropriate.
cDNA Microarray Technology
One of the major technical merits of microarray analysis is
its high sensitivity in the detection of mRNAs. Sensitivity,
Figure 3. Comparison of cDNA Microarray and RNA Gel Blot Analy-
sis for New DREB1A Target Genes and the DREB1A Gene.
Samples from dehydrated (dry 2 hr or dry 10 hr), cold-treated (cold 2
hr or cold 10 hr), or untreated 35S:DREB1A transgenic plants
(35S:DREB1A control) were fluorescently labeled with Cy3-dUTP,
and samples from untreated wild-type (WT) plants (control) were la-
beled with Cy5-dUTP. After hybridization with a cDNA microarra
y
and scanning, relative expression ratios were calculated and are in-
dicated below the RNA gel blots. The full-length cDNA sequences o
f
three DREB1A target genes (FL3-5A3, FL3-27, and FL5-2I22) have
been submitted to the GenBank, EMBL, and DDBJ databases with
accession numbers AB044404, AB044405, and AB046991 respec-
tively. h, hour; n.d., not determined.
68 The Plant Cell
which is determined by the minimal signal that can be reli-
ably detected above the background, is dependent in part
on background levels and on the specific incorporation of
label into the probes. EST microarrays have been reported
to detect 1:100,000 (w/w) (Ruan et al., 1998) or 1:500,000
(w/w) (Schena et al., 1996) of the total mRNAs. In our sys-
tem, 3.3 pg out of 1 g of poly(A)
⫹
RNA (1:300,000) could be
detected. These levels are generally considered sufficient to
detect an mRNA present at a few copies per cell (Ruan et
al., 1998). In this study, we prepared the probes from 1 g
of poly(A)
⫹
RNA. The original methods for preparing a probe
required ⬎1 g of poly(A)
⫹
RNA (Eisen and Brown, 1999);
however, it is difficult to obtain much mRNA from special-
ized cell types. The method for amplification of RNA isolated
from a single cell has been reported (Eberwine et al., 1992).
This method may extend the utility of microarray analysis of
tissue-specific gene expression.
In the present study, we used Imagene version 2.0 (Bio-
Discovery, Inc., Los Angeles, CA) and QuantArray (GSI Lu-
monics, Watertown, MA) as microarray data analysis
software. Companies that provide microarrays often provide
the necessary software and information to examine the ex-
perimental data (Richmond and Somerville, 2000). Scanning
and image processing currently require human intervention
to ensure that grids are properly aligned and that artifacts
are flagged and excluded from subsequent analysis. Adop-
tion of standard input/output formats, automation of feature
identification, and software identification of common arti-
facts are important goals for the next generation of microar-
ray analysis software. The ideal image analysis software
package should detect automatically each valid spot on the
array, flag and exclude bad spots, and subtract the local
background (Bassett et al., 1999). Also, genomic information
resources can be highly synergistic, and public databases
and tools such as GenBank, ENTREZ, and BLAST provide bi-
ologists with integrated and linked information. A GenBank-
like public database of gene expression measurements, inte-
grated with MEDLINE, ENTREZ, and other data and tools,
would be a useful resource for the biological community.
Conclusions and Perspectives
In the present study, we identified 44 drought-inducible
genes and 19 cold-inducible genes using the full-length
cDNA microarray (Tables 1 and 2). Among them, 30 and 10
Figure 4. Classification of the Drought- or Cold-Inducible Genes Identified into Four Groups on the Basis of RNA Gel Blot and Microarray Anal-
yses.
The drought- or cold-inducible genes identified were grouped into the following three groups: (1) drought- and cold-inducible genes, (2) drought-
specific inducible genes, and (3) cold-specific inducible genes. The following 21 genes were not grouped because of the difficulty of grouping
them into the three groups: erd3, erd14, rd19A, rd22, FL5-1F23, FL3-5J1, FL5-1N11, FL5-2H15, FL5-2G21, FL5-2I23, FL5-1C20, FL2-1H6, FL5-
2E17, FL3-3B1, FL5-3E18, FL3-2C6, FL5-1P10, FL2-5G7, FL2-1C1, FL2-5A4, and FL5-3P12. As a result, they were grouped as 20 drought- and
cold-inducible genes, five drought-specific inducible genes, and two cold-specific inducible genes. Drought- and cold-inducible genes were
classified into two groups: (1) DREB1A target genes and (2) non-DREB1A target genes. Sixteen drought- and cold-inducible genes were
grouped as 12 DREB1A target genes and four non-DREB1A target genes. Four drought- and cold-inducible genes (FL5-95, FL5-1O3, FL1-159,
and erd7) are not shown because (1) the expression level of three genes (FL5-95, FL5-1O3, and FL1-159) was less than twofold in 35S:DREB1A
transgenic plants than in wild-type control plants or not determined in the cDNA microarray analysis, and (2) it was difficult to group erd7 into
DREB1A target genes or non-DREB1A target genes.
Arabidopsis Full-Length cDNA Microarray 69
genes are novel drought- and cold-inducible genes, respec-
tively. Furthermore, we identified 12 DREB1A target genes,
six of which are novel genes. These results indicate that full-
length cDNA microarray analysis is a powerful tool for the
identification of stress-inducible genes and target genes of
transcription factors that control stress-inducible gene ex-
pression. Using our full-length cDNA microarray, it is easy to
isolate full-length cDNAs for further functional analysis. We are
planning to isolate ⬎10,000 Arabidopsis full-length cDNAs
and prepare the cDNA microarrays using the cDNA clones
for the identification of new stress-inducible genes and
new target genes of stress-related transcription factors.
Furthermore, we will apply this cDNA microarray analysis to
identify plant hormone-inducible genes, tissue-specific
genes, and target genes of transcription factors.
Full-length cDNA microarray analysis is not only a method
for the systematic analysis of quantitative gene expression
but also an extremely powerful tool with which to find novel
genes that are expressed in certain conditions or in certain
tissues. Hybridization of cDNA microarrays containing com-
plete sets of genes may eventually replace classic differen-
tial screening and display procedures. This method can
provide new markers for various physiological processes in-
volved in abiotic stress responses, hormonal signal trans-
duction, pathogen attack, and developmental processes. It
can also be used for the characterization of the molecular
basis of phenotypic changes in various mutants and trans-
genic plants and for the study of genetic networks through
the analysis of epistatic relationships between mutant phe-
notypes. Functional analysis, by use of parallel expression
monitoring, should help researchers to better understand
the fundamental mechanisms that underlie plant growth and
development. Microarray analysis using full-length cDNAs
provides a means to link genomic sequence information and
functional analysis. By accumulating data on gene expres-
sion by tissue type, developmental stage, hormone and her-
bicide treatment, genetic background, and environmental
conditions, it should be possible to identify the genes in-
volved in many important processes of development and re-
sponses to environmental conditions in plants.
Table 3. ABRE, DRE, and CCGAC Core Sequences Observed in the Promoter Regions of the DREB1A Target Genes Identified by cDNA
Microarray Analysis
a
Gene ABRE (PyACGTG (T/G) C) DRE (TACCGACAT) CCGAC Core Motif (CCGAC)
rd29A TACGTGTC (⫺45 to ⫺38)
b
TACCGACAT (⫺148 to ⫺140) AGCCGACAC (⫺111 to ⫺103)
TACCGACAT (⫺206 to ⫺198) GACCGACTA (⫺256 to ⫺248)
cor15a CACGTGGC (⫺132 to ⫺125) — GGCCGACCT (⫺184 to ⫺176)
GGCCGACAT (⫺361 to ⫺353)
AACCGACAA (⫺416 to ⫺424)
kin1 — TACCGACAT (⫺120 to ⫺112) ATCCGACAT (⫺720 to ⫺712)
kin2 CACGTGGC (⫺68 to ⫺61) TACCGACAT (⫺127 to ⫺119) CCCCGACGC (⫺403 to ⫺395)
rd17 TACGTGTC (⫺920 to ⫺913) — TACCGACTT (⫺162 to ⫺154)
AGCCGACCA (⫺967 to ⫺959)
GACCGACAT (⫺997 to ⫺989)
erd10 CACGTGGC (⫺838 to ⫺831) — GACCGACGT (⫺198 to ⫺190)
c
GACCGACCG (⫺202 to ⫺194)
c
CACCGACCG (⫺206 to ⫺198)
c
GACCGACAT (⫺999 to ⫺991)
FL3-5A3 CACGTGGC (⫺74 to ⫺67) TACCGACAT (⫺415 to ⫺407) TGCCGACAT (⫺806 to ⫺798)
FL3-27 — TACCGACAT (⫺89 to ⫺91) —
FL5-2122 — — TACCGACTC (⫺191 to ⫺183)
TACCGACTA (⫺266 to ⫺258)
TGCCGACCT (⫺418 to ⫺410)
ACCCGACTA (⫺695 to ⫺703)
GACCGACGT (⫺786 to ⫺778)
FL5-77 — — CCCCGACTA (⫺315 to ⫺307)
FL5-94 — — TACCGACTA (⫺190 to ⫺198)
TTCCGACTA (⫺260 to ⫺268)
ATCCGACGC (⫺630 to ⫺622)
a
ABRE, DRE, and CCGAC core sequences observed in 1000-bp upstream regions of the 5⬘ termini of the longest cDNA clones isolated are listed.
b
Numbers in parentheses indicate the nucleotide beginning at the 5⬘ terminus of the longest cDNA clone isolated. Minus signs indicate that the
nucleotide exists upstream of the 5⬘ terminus of the putative transcription start site.
c
These 9-bp sequences that contain a CCGAC core motif overlap each other.
70 The Plant Cell
METHODS
cDNA Clones
In the cDNA microarray analyses, we used ⵑ1300 cDNA sequences
representing Arabidopsis thaliana full-length cDNA clones isolated
from full-length cDNA libraries, the drought- and cold-inducible
genes responsive to dehydration (rd) and early responsive to dehy-
dration (erd), kin1, kin2, and cor15a, and the ␣-tubulin gene as an in-
ternal control. As a negative control, two DNAs derived from the
mouse nicotinic acetylcholine receptor epsilon-subunit (nAChRE) gene
and the mouse glucocorticoid receptor homolog gene were used.
Sequence Analysis
Plasmid DNA was extracted with a Kurabo DNA extraction instru-
ment (model PI-100; Kurabo, Tokyo, Japan) and subjected to se-
quencing. DNA sequences were determined using the dye terminator
cycle sequencing method with a DNA sequencer (model ABI Prism
3700; Perkin-Elmer Applied Biosystems, Foster City, CA). Sequence
homologies were examined with the GenBank/EMBL database using
the BLAST program.
Amplification of cDNA Inserts
The vector used for cDNA library construction was ZAPII (Carninci
et al., 1996). Inserts of cDNA clones were amplified by polymerase
chain reaction (PCR) using primers that were complementary to vec-
tor sequences flanking both sides of the cDNA insert. The primers
were 5⬘-CGCCAGGGTTTTCCCAGTCACGA (FL forward 1224
primer) and 5⬘-AGCGGATAACAATTTCACACAGGA (FL reverse 1233
primer). Plasmid templates (1 to 2 ng) were added to 100 L of a
PCR mixture containing 0.25 mM each nucleotide, 0.2 M each
primer, 1 ⫻ Ex Taq buffer (Takara Shuzo, Kyoto, Japan), and 1.25
units of Ex Taq polymerase (Takara Shuzo). PCR was performed as
follows: at 94⬚C for 3 min; for 35 cycles at 95⬚C for 1 min, 60⬚C for 30
sec, and 72⬚C for 3 min; and at 72⬚C for 3 min. To clean up PCR
products and prepare the DNA for printing, we precipitated PCR
products in ethanol and resuspended the DNA in 25 L of 3 ⫻ SSC
(1 ⫻ SSC is 0.15 M NaCl and 0.015 M sodium citrate). One microliter
of each finished reaction was electrophoresed on a 0.7% agarose
gel to confirm amplification quality and quantity.
cDNA Microarray Preparation
PCR products were arrayed from 384-well microtiter plates onto
poly-
L-lysine–coated micro slide glass (model S7444; Matsunami,
Osaka, Japan) using the GTMASS System gene tip microarray
stamping machine (Nippon Laser and Electronics Laboratory,
Nagoya, Japan). The tip loaded 0.5 L of PCR products (100 to 500
ng/L) from 384-well microtiter plates and deposited 5 nL per slide
on six slides with spacing of 280 m. The printed slides were rehy-
drated in a beaker with hot distilled water and snap dried at 100⬚C for
3 sec. The slides were placed into a slide rack, and the rack was
placed into a glass chamber. The blocking solution, containing 15
mL of 1 M sodium borate, pH 8.0, 5.5 g of succinic anhydride (Wako,
Osaka, Japan), and 335 mL of 1-methyl-2-pyrrolidone (Wako), was
poured into the glass chamber. The slide racks were plunged up and
down five times, shaken gently for 15 min, transferred into a chamber
containing boiling water, plunged up and down another five times,
and left for 2 min. The slide racks were transferred into a chamber
containing 95% ethanol, plunged up and down five times, and cen-
trifuged at 2500g for 30 sec.
Plant Materials and RNA Isolation
Wild-type Columbia plants and transgenic plants overexpressing
DREB1A cDNA under the control of the cauliflower mosaic virus
(CaMV) 35S promoter (Kasuga et al., 1999) were grown on germina-
tion medium agar plates for 3 weeks as described previously
(Yamaguchi-Shinozaki and Shinozaki, 1994). Dehydration and cold
stress treatments were performed as described previously (Yamaguchi-
Shinozaki and Shinozaki, 1994). The Columbia plants were subjected
to stress treatments for 2 or 10 hr and then frozen in liquid nitrogen
for further analysis. In the experiments to identify DREB1A target
genes, transgenic plants overexpressing DREB1A cDNA and wild-
type plants grown on germination medium lacking kanamycin were
used. Transgenic plants overexpressing DREB1A cDNA were not
subjected to stress treatment. Total RNA was prepared using Isogen
(Nippon Gene, Tokyo, Japan), and mRNA was prepared using the
Oligotex-dT30 mRNA purification kit (Takara, Tokyo, Japan).
Preparation of Fluorescent Probes
Each mRNA sample was reverse transcribed in the presence of Cy3
dUTP or Cy5 dUTP (Amersham Pharmacia). The reverse transcrip-
tion reaction was performed in a 30-L volume containing 1 g of
poly(A)
⫹
RNA with 6 g of oligo(dT) 18mer, 10 mM DTT, 500 M each
dATP, dCTP, and dGTP, 200 M dTTP, 100 M Cy3 dUTP or Cy5
dUTP, and 400 units of SuperScript II reverse transcriptase (Life
Technologies, Grand Island, NY) in 1 ⫻ Superscript first-strand
buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl
2
, and 20 mM
DTT) (Life Technologies). After incubation at 42⬚C for 1 hr, the reac-
tion products of two samples (one with Cy3 labeling and the other
with Cy5 labeling) were combined, treated with 15 L of 0.1 M NaOH
and 1.5 L of 20 mM EDTA, incubated at 70⬚C for 10 min, and
treated with 15 L of 0.1 M HCl. The samples were placed in a Mi-
crocon 30 microconcentrator (Amicon, Beverly, MA). Four hundred
microliters of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA)
was added and spun for 5 to 10 min in a benchtop microcentrifuge at
a high speed to a volume of 10 to 20 L. The flow-through product
was discarded. Four hundred microliters of TE buffer and 20 L of 1
mg/mL human Cot-1 DNA (Gibco BRL) were added and spun again.
After the second spin, the probe retained by the Microcon was sig-
nificantly brighter than the flow-through product. This is a very strong
indicator of a successful labeling reaction. The probes were col-
lected by inverting the filter and spun for 5 min. Several microliters of
distilled water was added to the Microcon. The filter was inverted
and spun so that the final volume of the collected probes was 12 L.
Two microliters of 10 g/L yeast tRNA, 2 L of 1 g/L pd(A)
12–18
(Amersham Pharmacia), 3.4 L of 20 ⫻ SSC, and 0.6 L of 10% SDS
were added to the probes. The probe samples were denatured by
placing them in a 100⬚C water bath for 1 min, left at room tempera-
ture for 30 min, and then used for hybridization.
Arabidopsis Full-Length cDNA Microarray 71
Microarray Hybridization and Scanning
The probe samples were spun for 1 min in a benchtop microcentri-
fuge at high speed to pellet any particulate matter. The probes were
placed onto the center of the array to avoid forming bubbles. A cover
slip was placed over the entire array surface to avoid the formation of
bubbles. Four 5-L drops of 3 ⫻ SSC were placed on a separate part
of the slide to provide humidity in the hybridization chamber and thus
ensure that the probe mixture did not dehydrate during hybridization.
The slides were placed in a sealed hybridization cassette (model
THC-1; BM Equipment, Tokyo, Japan) and submerged in a 65⬚C wa-
ter bath for 12 to 16 hr. After hybridization, the outside of the slide
chamber was dried carefully. The slides were removed and placed in
a slide rack submerged in washing solution 1 (2 ⫻ SSC, 0.1% SDS),
with the array face of the slide tilted down so that when the cover slip
dropped off it did not scratch the array surface. After the cover slip
came off, the slide racks were plunged up and down three times in
washing solution 1 and transferred to washing solution 2 (1 ⫻ SSC)
carefully to minimize the transfer of washing solution 1 to the second
chamber, because SDS can interfere with slide imaging. The slide
chamber was rocked gently for 2 min. The slide racks were trans-
ferred to washing solution 3 (0.2 ⫻ SSC), allowed to stand for 2 min,
spun at 2500g for 1 min, and dried.
Microarrays were scanned with a scanning laser microscope
(model ScanArray4000; GSI Lumonics). Separate images were ac-
quired for each fluor at a resolution of 10 m per pixel. To normalize
the two channels with respect to signal intensity, we adjusted photo-
multiplier and laser power settings so that the signal ratio of the ␣-tubu-
lin genes (internal control) was as close to 1.0 as possible.
For the microarray data analysis, we used Imagene version 2.0
(BioDiscovery) and QuantArray (GSI Lumonics) software.
RNA Gel Blot Analysis
Isolated total RNA was also used for RNA gel blot hybridization.
Hybridization was performed as described previously (Yamaguchi-
Shinozaki and Shinozaki, 1994). The PCR-amplified fragments pre-
pared from the full-length Arabidopsis cDNAs were used as probes
for RNA gel blot hybridization.
ACKNOWLEDGMENTS
We thank Suzurei Shimamura, Masahiro Yonezawa, Suguru
Okunuki, Setsuko Kawamura, Mihoko Ohsaki, and Shigeko Nagano
for their skilled technical assistance. We thank Dr. Yasushi Okazaki
and Rika Miki for technical advice. We thank Nippon Laser and Elec-
tronics Laboratory, BM Equipment Co., Ltd., General Scanning, Inc.,
and Dr. Takaaki Sato for technical support. We thank Dr. D.P. Snustad
for the gift of the ␣-tubulin gene. This work was supported in part by
a grant for genome research from RIKEN, the Program for the Pro-
motion of Basic Research Activities for Innovative Biosciences, the
Special Coordination Fund of the Science and Technology Agency,
and a grant-in-aid from the Ministry of Education, Science, and Cul-
ture of Japan to K.S. It was also supported by a Grant-in-Aid for
Scientific Research on Priority Areas (C) “Genome Science” from
the Ministry of Education, Science, and Culture of Japan to M.S. It
was also supported in part by the Core Research for Evolutional Sci-
ence and Technology program of the Japan Science and
Technology Corporation, the Special Coordination Fund, a research
grant for the Genome Exploration Research Project from the Sci-
ence and Technology Agency, and a Grant-in-Aid for Scientific
Research on Priority Areas and the Human Genome Program from
the Ministry of Education and Culture, Japan, to Y.H.
Received August 10, 2000; accepted November 13, 2000.
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