A bZIP transcription factor, OsABI5, is involved in rice fertility

and stress tolerance

Meijuan Zou Yucheng Guan Haibo Ren
Fang Zhang Fan Chen
Received: 21 August 2007 / Accepted: 16 January 2008 / Published online: 31 January 2008
Springer Science+Business Media B.V. 2008
Abstract The phytohormone abscisic acid (ABA) is
involved in the adaptive stress response and regulates
expression of many stress-responsive genes, including
some transcriptional factors. A bZIP transcription factor,
OsABI5, was isolated from the panicle of Oryza sativa L.
Expression of the OsABI5 gene was induced by abscisic
acid (ABA) and high salinity, and down-regulated by
drought and cold (4C) in seedlings. The OsABI5 protein
was localized in the nucleus and has trans-activation
activity. The N-terminal of the protein is necessary for its
activity. OsABI5 could bind to a G-box element and trans-
activate reporter gene expression. Complementation anal-
ysis revealed that the expression of OsABI5 driven by the
35S promoter could rescue ABA-insensitivity of abi5-1
during seed germination and result in hypersensitivity to
ABA. Over-expression of OsABI5 in rice conferred high
sensitivity to salt stress. Repression of OsABI5 promoted
stress tolerance and resulted in low fertility of rice. These
results suggested that OsABI5 could regulate the adaptive
stress response and plant fertility of rice as a transcription
factor.
Keywords ABA bZIP transcription factor OsABI5
Plant fertility Stress endurance Transgenic rice
Abbreviations
ABA Abscisic acid
abi3 Abscisic acid insensitive 3
abi5 Abscisic acid insensitive 5
Introduction
Throughout plant development, the phytohormone abscisic
acid (ABA) plays a crucial role in the adaptive response to
abiotic stresses such as drought, cold and high salinity. It is
also involved in various aspects of plant growth including
seed maturation, dormancy, inhibition of cell division and
germination (Leung and Giraudat 1998). During seed
maturation in many species, ABA gradually accumulates
and triggers expression of many stress-responsive genes. In
plants, several transcription factors have been identied that
control various aspects of seed maturation and germination
(Shiota et al. 1998; Carrari et al. 2001; Nambara et al. 2000;
Casaretto and Ho 2003). The abi5 locus was identied by
screening for decreased sensitivity to ABA inhibition of
germination in Arabidopsis thaliana (Finkelstein and Lynch
2000). The AtABI5 gene encodes a basic leucine zipper
transcription factor. Mutation of the gene resulted in pleio-
tropic defects in the response to ABA, including altered
expression of some ABA-regulated genes. ABI5 and its
homologs physically interact with other ABA-responsive
transcription activators, ABI3 or its orthologs, to regulate
seed-specic and/or ABA-inducible gene expression
(Lopez-Molina et al. 2002; Nakamura et al. 2001; Hobo
et al. 1999; Casaretto and Ho 2003). Genetic studies showed
that the ABI5 gene, together with ABI3, may be involved in
ABA signal transduction as an important regulator during
seed maturation and germination.
OsABI5: GenBank accession No. EF199631.
M. Zou Y. Guan H. Ren F. Zhang F. Chen (&)
Key Laboratory of Molecular and Developmental Biology,
National Centre for Plant Gene Research, Institute of Genetics
and Developmental Biology, Chinese Academy of Sciences,
P.O. Box 2707, South 1-3, Zhongguancun, Beijing 100080,
P.R. China
e-mail: fchen@genetics.ac.cn
1 3
Plant Mol Biol (2008) 66:675683
DOI 10.1007/s11103-008-9298-4
The bZIP-type transcriptional factors are involved in
developmental and physiological processes in response to
stresses, and are important for various plants to withstand
adverse environmental conditions (Uno et al. 2000; Jakoby
et al. 2002). As transcription factors, they may interact with
specic ABA-responsive cis-acting elements (ABRE) and
trans-activate down-stream gene expression. Using various
promoter analyses, the G-box element 5
0
CACGTG3
0
(ABRE) has been shown to be present in many stress-
responsive genes and is involved in ABA-regulated expres-
sion (Niu et al. 1999; Kimet al. 1998; Martinez-Garcia et al.
1998; Siberil et al. 2001). A number of ABRE-binding fac-
tors have been isolated from different plants (Carles et al.
2002; Niu et al. 1999; Choi et al. 2000; Kang et al. 2002).
AtABI5 belongs to a subfamily of bZIP transcriptional fac-
tors (Finkelstein and Lynch 2000) and strongly binds to
ABRE in the AtEm6 promoter (Carles et al. 2002).
Rice is animportant economic cropandfeeds manypeople.
Understanding the mechanisms of stress responses in rice will
provide a fundamental foundation for research on plant
endurance. At present, most reports on stress responses focus
on Arabidopsis. Redundant and distinct functions of the ABA
response loci ABI5 and ABF3 have also been found in Ara-
bidopsis (Finkelstein et al. 2005). The conservation and
specicity of the ABAsignaling pathway and stress responses
in rice compared with Arabidopsis remains largely unknown.
In this paper, we present the isolation and identication of a
rice bZIP transcription factor, OsABI5. It displayed high
homology with the Arabidopsis AtABI5 gene, suggesting
similar functions in the adaptive stress response and the ABA
signaling pathway. We analyzed the biological function of
OsABI5 in vivo using transgenic plants. Tolerance to salt
stress and fertility of rice were affected by decreasing
expression of OsABI5. OsABI5 may have overlapping and
distinct functions with AtABI5 during plant development.
Materials and methods
Plant materials and growth conditions
The wild-type rice seeds (Oryza sativa L.) were immersed in
water for 1 day and grown for 1 week in a greenhouse under
controlledconditions (16 hlight/8 hdarkcycle) at 27C. One-
week-old seedlings were used in various stress treatments.
The Arabidopsis seedlings of wild-type Wassilewskija
(Ws-2), abi5-1 mutants, and transgenic plants were plated on
1/2 MS supplemented with different concentrations of ABA,
chilled for 2 days at 4C in darkness, and incubated for 3 or
12 days at 22C with a 16 h photoperiod to compare the
germination rates.
The variety of rice used for transformation experiments
was Nongken 58 (O. sativa L. ssp. japanica), provided by
the Institute of Agriculture Science of HuBei, P.R.China.
The transgenic lines and control were grown under con-
trolled conditions.
The seeds of wild-type Ws-2 (stock number CS22659)
and strain abi5-1 (stock number CS8105), which has a
mutation in the AtABI5 gene (AT2G36270), were obtained
from the Arabidopsis Biological Resource Center (ABRC)
at Ohio State University (Columbus, OH, USA).
RT-PCR analysis
Total RNAs were isolated from 10-day-old seedlings that
had been treated with phytohormones and/or stress, using
Trizol reagent (Gibco). Reverse transcription-PCR was
performed according to the manufacturers instructions
(SuperScript. II RNase H
-
Reverse Transcriptase, Invitro-
gen). Primers 5
0
-ATGGCATCGGAGATGAGCAAGAA
C-3
0
and 5
0
-GCTTCTTTGTCAGTAGAACCGTCTTC-3
0
were usedtotest the expressionpatternof OsABI5inseedlings
under different treatments and in transgenic lines. Total RNAs
were isolated from the young panicle in rice transgenic lines
to analyze gene expression. The actin gene was amplied as
an internal control to quantify the relative amounts of cDNA.
Subcellular localization of OsABI5::GFP in onion
epidermal cells
We amplied the OsABI5 cDNA with the primers containing
two attB recombination sites, subcloned the products into a
donor vector by recombination in vitro (Gateway BP Clonase
Enzyme Mix, Invitrogen) and created an entry vector. The LR
clonase reaction to transfer DNAfragments fromentry clones
to destination vectors pMDC83 was carried out according to
the manufacturers instructions (Invitrogen). The OsA-
BI5::GFP fusion protein driven by the CaMV 35S promoter
was introduced into onion epidermal cells with an Agrobac-
terium-mediated system, incubated on 1/2 MS medium for
24 h at 26C in darkness, and the uorescence of GFP was
visualized under a uorescence microscope.
Transactivation analysis in yeast
The trans-activation assay was performed according to the
methods described by Zou et al. (2007). The yeast stains
AH109 and Y187 harboring the LacZ and HIS3 reporter
genes were used as an assay system (Clontech) to examine
the gene for the presence of an activation domain. The
complete and partial regions of the OsABI5 cDNA were
cloned into the DNA-binding domain vector pGBKT7
(Clontech). The plates were incubated for 3 days, and
676 Plant Mol Biol (2008) 66:675683
1 3
analyzed by b-galactosidase assays. A colony-lift lter
assay was carried out according to the yeast handbook
instructions (Clontech).
DNA binding assay
We constructed a yeast reporter strain that carried the dual
reporter genes HIS3 and lacZ, with a trimer of 27 bp DNA
fragments upstream of the TATA element composed as
follows:
5
0
-agctAGCCACGTGTCGGACACGTGGCA-3
0
3
0
-TCGGTGCACAGCCTGTGCACCGTtcga-5
0
The 27 bp fragment contained two G-box motifs and a
5
0
-agct-3
0
annealing end, which was ligated into three
tandemly repeated copies and then inserted into the HindIII
site in the multicloning site (MCS) of the pBluescript II SK
+
(Stratagene) vector. The fragment containing three tandem
copies of the 27 bp was excised by EcoRI and BssHII from
the pBluescript II SK
+
vector. It was then cloned into the
multicloning site (MCS) upstream from the HIS3 minimal
promoter in the pHISi-1 expression vector (Clontech), which
had been digested with EcoRI and MluI. The same fragment
was excised by EcoRI and SalI from the pBluescript II SK
+
vector and cloned into the MCS upstream from the lacZ
minimal promoter in the pLacZi expression vector (Clon-
tech), which had been digested with the same enzymes. Two
kinds of expression plasmids were transformed simulta-
neously into the yeast strain YM4271 (Clontech). Yeast
transformants containing the HIS3 and lacZ reporter genes
were obtained using selective medium plates (without His
and Ura). The resulting yeast strain transcribed the HIS3 gene
at basal levels, growing on media lacking histidine (but not in
the presence of 30 mM 3-aminotriazole [3-AT], a compet-
itive inhibitor of the HIS3 gene product), and formed white
colonies on lter papers containing X-gal. These yeast cells
were separately transformed with the yeast expression vector
pAD-Gal4-2.1, which harbored the OsABI5 genes. The plates
were incubated for 3 days, and then analyzed by colony-lift
lter assay according to the yeast handbook instructions
(Clontech). If the fusion protein interacted with the G-box
elements, HIS3 reporter gene expression was activated,
allowing colony growth on minimal medium lacking histi-
dine with 3-AT. The 3-ATresistant yeast strains also
induced lacZ activity and formed blue colonies.
Construction of OsABI5 transgenic plants and stress
treatments
The cDNA of OsABI5 was cloned into the vector pCAM-
BIA1301 driven by the CaMV 35S promoter, resulting in
the construct 35S::OsABI5(+). All constructs were veried
by sequencing. The Arabidopsis transgenic lines (Ws-2 and
abi5-1) were generated by the Agrobacterium tumefaciens
oral-dip method (Clough 1998). Seeds (T1) from inl-
trated plants were plated on 1/2 MS medium containing
25 lg l
-1
hygromycin (Roche). The homologous T3 gen-
eration seeds were analyzed.
The cDNA of OsABI5 was inserted in the reverse ori-
entation into the pCAMBIA1301 vector, resulting in the
antisense construct 35S::OsABI5(-). The 35S::OsABI5(+)
and 35S::OsABI5(-) constructs were introduced into
Nonken 58 (O. sativa L.) by an Agrobacterium-mediated
system, and cultured on medium containing 50 lg l
-1
hygromycin (Roche). The 20-day-old seedlings of T1
generation transgenic plants were used to test stress sen-
sitivity under 250 mM NaCl treatment for 25 days, or 15%
PEG treatment for 5 days. The growth status was observed.
Pollen analysis
Pollen grains from the antisense and control plants were
collected and stained with a 1% I
2
KI solution to observe
starch accumulation. Stained pollen grains were examined
directly under a microscope and photographed.
Results
Isolation of bZIP transcription factor OsABI5 from rice
A 1416 bp cDNA clone was predicted by TIGR Rice Gen-
ome Annotation Database (http://www.tigr.org) and isolated
from the young panicle of rice by RT-PCR. It shares high
amino acid sequence homology with Arabidopsis AtABI5
and barley HvABI5 within ve regions (Genetyx 5.0 soft-
ware) (Fig. 1) and was designated as OsABI5 (GenBank
accession No. EF199631). Amino acids homology analysis
showed that the OsABI5-encoded peptide contains a typical
basic leucine zipper domain. The deduced amino acid
sequence of OsABI5 contains conserved regions similar to
other ABI5 or ABI5-like genes in other species. The Arabid-
opsis AtABI5 gene is involved in seed dormancy, maturation
and stress responses (Brocard et al. 2002). The barley HvA-
BI5 is responsible for ABA-induced gene expression
(Casaretto et al. 2003). The conservation of some functional
domains described above may predict functional similarities
during plant development and stress responses.
Expression patterns of the OsABI5 gene
Expression patterns of the OsABI5 gene under various
environmental stresses and ABA treatment were
Plant Mol Biol (2008) 66:675683 677
1 3
analyzed by RT-PCR. As shown in Fig. 2, OsABI5 was
induced within 1 h after ABA and high-salt treatment,
and the mRNA level continuously increased up to 24 h.
Cold treatment (4C) initially suppressed OsABI5
expression within 5 h, and then induced it to reach its
maximum at 24 h. When plants were subjected to
dehydration stress, OsABI5 expression was suppressed
within 24 h.
Subcellular localization of OsABI5-GFP fusion protein
A basic region containing a nuclear localization signal was
found in OsABI5. To examine the localization of the
OsABI5 protein, we transiently expressed OsABI5 in the
epidermal cells of onion. Protein expression was observed
under a uorescence microscope. The result showed that
the OsABI5::GFP fusion protein was targeted to the nuclei
Fig. 1 Comparison of the
deduced amino acids of OsABI5
with other bZIP proteins.
Multiple sequence alignment of
the OsABI5 amino acid
sequence with other bZIP
protein sequences, TRAB1
(GenBank accession No.
BAA83740), HvABI5
(GenBank accession No.
AY150676), and ABI5
(GenBank accession No.
AAD21438). Blast analysis was
conducted with Genetyx
software (5.0). The identical
residues are shaded in black.
The basic domain and leucine
repeats of the bZIP domain are
double-underlined and
conserved regions are single-
underlined. Asterisks (*) denote
the proline-rich (P rich) region
Cold
Drought
Salt
ABA
actin
0 1 2 5 10 24 h
Fig. 2 RT-PCR analysis of OsABI5 expression patterns under ABA
and stress conditions. Total RNAs were isolated from 7-day-old rice
seedlings treated with 100 lM ABA, cold (4C), drought, and
250 mM NaCl at the indicated times. The length of PCR products is
1140 bp. The rice actin gene was used as an internal control
Fig. 3 Nuclear localization of the OsABI5 protein. OsABI5::GFP
fusion protein was expressed transiently under the control of CaMV
35S promoter in onion epidermal cells and observed under a
uorescence microscope. The photographs were taken in dark eld
for green uorescence (b), in bright light for the morphology of the
cell (a), and in combination (c)
678 Plant Mol Biol (2008) 66:675683
1 3
of the cells (Fig. 3). These data suggested that OsABI5 was
a nuclear protein and functioned as a transcription factor to
regulate expression of down-stream genes.
Transactivation activity and DNA-binding activity
of OsABI5 to the cis-acting G-box element in yeast
A yeast one-hybrid system was used to determine the
transcription activation activity of OsABI5. The entire
coding region and several N-terminal regions of OsABI5
with partial deletions were fused to the GAL4 DNA-
binding domain (Fig. 4a). These constructs were trans-
formed into yeast (Y187) harboring GAL4-binding sites
fused upstream of a lacZ reporter gene, and the growth
status of transformants was observed. All of these yeast
cells grew well on YPAD medium (Fig. 4c).The yeast cells
containing pBD-OsABI5 and the positive control grew well
on SD medium without histidine, but the others could not
grow on selection medium (Fig. 4d). A colony-lift lter
assay supported these results (Fig. 4e). These results showed
that the full-length OsABI5 had obvious activation capability.
However, no activation activity was observed in strains
where the construct contained a deletion of 80 amino acids at
the N-terminal coupled with an intact C-terminal portion
containing the bZIP domain. This indicated the necessity of
the N-terminal region for transactivation activity.
The bZIP protein is known to interact mainly with DNA
cis-elements containing a 5
0
-ACGT-3
0
core sequence. The
binding specicity of OsABI5 to the G-box element was
determined by a yeast one-hybrid system (Zou et al. 2007).
The results indicated that OsABI5 can function as a trans-
acting factor for the G-box element.
Suppression of the OsABI5 gene by antisense RNA
causes aberrant pollens and low fertility of rice
To investigate the possible function of the OsABI5 gene
during rice development, the antisense OsABI5 cDNA was
introduced into rice using an Agrobacterium-mediated
system. The positive transgenic lines were conrmed by
PCR amplication using primers from hygromycin (data
not shown). In total, 13 independent antisense transgenic
plants were obtained and analyzed. The average fertility
rate of the antisense lines was approximately 49.5%, while
the fertility rate of the control was approximately 93%. The
antisense plants all displayed low fertility (Fig. 5b). A
semi-quantitative RT-PCR analysis was used to detect the
OsABI5 mRNA expression in antisense lines and the cor-
responding fertility rates were evaluated. Plants carrying an
empty vector were controls. Results showed that the
expression of OsABI5 was suppressed to different degrees
and the decreased fertility rate of rice was correlated with
the expression of OsABI5 (Fig. 5a).
Microarray data showed that the OsABI5 gene was highly
expressed in the mature pollen, which suggests that it may
play an important role in pollen maturation (Lan et al. 2005).
Pollen grains were collected from the mature transformed
plants to investigate the pollen status. The pollen grains of the
control were stained uniformly using I
2
-KI staining. How-
ever, in transgenic lines more than 60% of microspores in
mature pollen showed an abnormal shape and did not stain,
which suggests they are sterile (Fig. 5c). Therefore we
speculated that suppression of the OsABI5 mRNAlevel might
cause abnormal development of mature pollen and low fer-
tility of transgenic lines of rice. Together, this suggested that
OsABI5 was involved in pollen maturation.
240
388
388
80
388
GBD::OsABI5-C2
UAS
UAS
( Yeast: GAL4,His3,Ura1 )
GBD::OsABI5
GBD::OsABI5-C1
bZIP
GAL4 BD
1
GAL4 BD
GAL4 BD
(A)
GBD::
OsABI5 OsABI5-C2
pGAL4
GBD::
GBD::
OsABI5-C1
pBD
YPDA Colony-lift Filter Assay SD/His
-
(B)
(C) (D)
(E)
Fig. 4 Trans-activation activity
of the OsABI5 protein. Fusion
proteins of the GAL4 DNA
binding domain and different
portions of OsABI5 were
expressed in yeast strain Y187.
The transformed yeast culture
was dropped onto YPDA or SD
plates without histidine and
growth status examined. The
plates were incubated for
3 days, and then analyzed by
b-galactosidase assay. pGAL4
and pBD were used as positive
and negative controls,
respectively
Plant Mol Biol (2008) 66:675683 679
1 3
Stress tolerance of OsABI5 transgenic plants
To examine the in vivo function of OsABI5 in stress
responses, we used plants over-expressing OsABI5, and
OsABI5-suppressed plants to investigate plant tolerance
to environmental stress. Expression of OsABI5 within
over-expression and suppression lines was also analyzed
(Fig. 5a, f). About 50 20-day-old T1 transgenic seedlings
at the same growth stage were exposed to 250 mM NaCl
for 25 days and 15% PEG for 5 days. We compared the
tolerance of OsABI5 over-expression plants and OsABI5-
suppressed plants to high salt concentration or PEG
treatment. The antisense OsABI5 transgenic plants grew
well in high salt and exhibited higher tolerance to salt,
which indicated that the suppression of OsABI5 expres-
sion increased salt tolerance. In contrast, higher
concentrations of NaCl caused visible wilting, and the
leaves of the 35S::OsABI5 transgenic lines showed
prominent chlorosis compared to the control (Fig. 5d).
Similarly, the antisense transgenic lines also displayed
high tolerance to the PEG treatment (Fig. 5e) and no
obvious difference was found between control and over-
expression lines in rice (data not shown). These results
suggested that the extent of tolerance to salt or PEG
stress of these plants is negatively correlated with the
expression level of OsABI5. Therefore, OsABI5 may be
involved in the stress tolerance of rice and may act as a
negative regulator.
Fig. 5 Analysis of the fertility rates, mature pollen and stress
tolerance of rice OsABI5 transgenic lines. 35S::OsABI5(+) and
35S::OsABI5(-) denote the OsABI5-over-expression and antisense
transgenic lines, respectively. (a) RT-PCR analysis of antisense
transgenic lines L2, L3, L6 and their fertility rates. RT-PCR was
performed for 37 cycles to investigate the OsABI5 expression. The
rice actin gene was used as an internal control. Plants carrying an
empty vector were controls. (b) Comparison of rice fertility status in
control and antisense transgenic lines. (c) Microspore status of mature
pollen stained by KI
2
(d) Stress tolerance of transgenic lines under
NaCl treatment. 20-day-old seedlings of rice transgenic lines were
exposed to 250 mM NaCl for 25 days. (e) Stress tolerance of
transgenic lines under PEG treatment. Seedlings (20-days-old) of the
control and antisense transgenic lines were exposed to 15% PEG for
5 days. (f) RT-PCR analysis of OsABI5-over-expression transgenic
lines. Lanes 1 and 2: rice over-expression transgenic lines. Plants
carrying an empty vector were controls. RT-PCR was performed for
30 cycles to investigate OsABI5 expression
680 Plant Mol Biol (2008) 66:675683
1 3
Effects on the expression of ABA-inducible
and stress-responsive genes
A number of genes have been reported to be induced by
drought, high-salinity, and low-temperature stresses, which
function in stress response and stress tolerance (Rabbani
et al. 2003; Ren et al. 2005). These genes are therefore good
markers of ABA- and stress responses throughout plant
development. Among these genes, Lip 9 (BP432938), aba45
(BP432968), salT (BP43300), and aba2 (BP432972) are
induced by drought, high salinity, and low temperature
(Rabbani et al. 2003). The OsLea3 and OsWsi18 genes,
encoding late-embryogenesis abundant proteins, both could
be induced by ABA or stress in seedlings, and function in
seed development (Moons et al. 1997; Joshee et al. 1998).
The SKC1 gene is a rice QTL gene encoding a sodium
transporter involved in the salt response (Ren et al. 2005).
Analyses of the expression of these stress-inducible genes in
transgenic lines can help to determine the possible regula-
tory relationships among OsABI5 and down-stream genes.
We compared the expression of these marker genes in the
antisense transgenic lines and vector controls at the panicle
stage. RT-PCR results showed that respressing OsABI5
expression altered the expression of aba45, aba2, SalT,
SKC1 and Wsi18. No change was found in the expression of
OsLea3, Lip9, OsEMP1 (Fig. 6). Interestingly, the Wsi18
gene has two transcripts (unpublished data) and their
expression was altered differently. The expression of SKC1
was decreased and SalT was increased in the antisense
transgenic lines with decreasing OsABI5 expression, indi-
cating the possible high tolerance to salt and a tight coupling
between stress tolerance and OsABI5 function. These results
suggest the existence of a complex regulatory relationship
between the transcription factor OsABI5 and the expression
of other genes.
Discussion
bZIP transcription factors play important roles in diverse
biological processes such as stress signaling, seed matu-
ration and ower development (reviewed by Finkelstein
et al. (2002)). In the present study, we reported the isola-
tion and characterization of a rice gene, OsABI5, which
belongs to a subfamily of bZIP transcription factors.
Amino acid sequence alignment demonstrated the high
homology to AtABI5, HvABI5, or an ABI5-like gene
(Fig. 1), indicating their functional similarity. The com-
plementation test further supported this. Combining the
results from gain-of-function and loss-of-function trans-
genic lines, we demonstrated that OsABI5 is involved in
the stress response and ABA signal transduction.
The OsABI5 protein was constitutively localized to the
nucleus. Other bZIP proteins of the ABI5 subfamily are
also constitutively located in the nucleus during embryo
maturation (Lopez-Molina et al. 2002; van der Krol and
Chua 1991; Bensmihen et al. 2005). We found that the
OsABI5 protein functions as a transcriptional activator in
yeast (Fig. 4). Deletion analysis showed that the N-termi-
nal region is necessary for transcriptional activation. These
data are consistent with the previous observations that the
other bZIP genes function as transcription activators
(Carles et al. 2002; Casaretto et al. 2003; Hobo et al.
1999). We further demonstrated that OsABI5 could bind to
the G-box element and activated the expression of reporter
genes. These results indicated that OsABI5 functioned as a
transcription factor to regulate the down-stream genes.
The expression of OsABI5 was induced by ABA and
stress treatment and may have an important physiological
function in plant development. To investigate the in vivo
functions of OsABI5, we generated Arabidopsis and rice
transgenic lines. Direct analysis of the responses of Ara-
bidopsis transgenic plants to exogenous ABA revealed that
the over-expression of OsABI5 conferred high sensitivity
to ABA (Zou et al. 2007). These ndings suggest that
OsABI5 functions as a transcriptional activator in ABA
signal transduction. A complementation test showed that
OsABI5 could rescue the ABA sensitivity of abi5-1, and
transgenic abi5-1 lines acquired ABA sensitivity similar to
SKC1
actin
Oslea3
Wsi18
SalT
Lip9
OsEMP1
aba45
aba2
L2 L3 L6 Control
Fig. 6 Comparative RT-PCR analysis of ABA or stress-responsive
gene expression in the different OsABI5 antisense transgenic lines.
The rice actin gene was used as an internal control. L2, L3, L6
denoted the different antisense lines. Plants carrying an empty vector
were controls
Plant Mol Biol (2008) 66:675683 681
1 3
WT during seed germination. Meanwhile, over-expression
of OsABI5 in WT Ws-2 resulted in the WT displaying
hypersensitivity to ABA inhibition of seed germination,
and a similar ABA response to plants with the AtABI5 gene
(Brocard et al. 2002). It has been suggested that ABI5 plays
a role in protecting germinating embryos from drought, and
ABI5 is regulated by ABA during germination (Lopez-
Molina et al. 2001). Based on the complementation
experiments and the similar phenotype of over-expression
transgenic lines, we propose that OsABI5 has a similar
function to AtABI5 in the ABA signal pathway.
The OsABI5 gene is also involved in the stress response.
The over-expression and repression of OsABI5 resulted in
opposite phenotypes in stress responses (Fig. 5d). The
seedlings of the OsABI5 over-expressed plants were sen-
sitive to salt stress, while the OsABI5-repressed plants
showed increased stress resistance to NaCl and PEG. This
result suggests that stress-responsive signaling is activated
in the OsABI5-repressed plants. The results show that
OsABI5 could prevent growth in a stressed environment.
This is probably because ABA, the level of which increases
under stress conditions, promotes the inhibition process.
SCK1, a rice quantitative trait locus for salt tolerance,
encodes a sodium transporter and is induced by ABA and
salt treatment (Ren et al. 2005). The expression of the salt-
responsive gene salT from rice is also regulated by ABA
and salt (Garcia et al. 1998) and is affected in the
35S::OsABI5(-) antisense plants (Fig. 6). This indicated a
tight coupling between stress tolerance and OsABI5 func-
tion. The expression of some ABA-inducible genes such as
aba2, aba45 and Wsi18, was affected by altered expression
of OsABI5 in the antisense transgenic lines. This suggested
that SalT, SCK1, OsABI5 and these ABA-inducible genes
may function in a common signaling pathway. It also
indicated that OsABI5 expression might be an important
mediator of these genes expression, and that OsABI5 is an
important determinant of ABA and salt signaling. The
expression of OsLea3, Lip9, and OsEMP1 was unchanged
in the antisense transgenic lines, which indicated that they
might function in a different ABA signaling pathway.
Microarray data had showed that the OsABI5 gene was
highly expressed in mature pollen (Lan et al. 2005), which
suggests that it may play an important role in pollen mat-
uration. Suppression of OsABI5 expression affected the
process of microspore formation in the antisense plants and
many abnormal microspores were observed. In all of the
antisense transgenic plants examined, pollen abortion
always appeared in mature pollen grains, which suggested
OsABI5 may be involved in the male gametes formation.
Meanwhile, the expression pattern of the OsABI5 gene on
the website (http://mpss.udel.edu/rice/) displayed a high
expression level in mature pollen, indicating that it may
modulate formation of mature pollen. ABA promotes
acquisition of desiccation tolerance during seed maturation
(Ooms Jaap et al. 1994). In Arabidopsis, the function
of AtABI5 genes in pollen development is unidentied.
AtABI5 is expressed in vegetative and oral organs and
regulates some genes correlated with desiccation tolerance
(Brocard et al. 2002; Swire-Clark and Marcotte 1999).
Whether AtABI5 plays a similar role in maturation of
anthers is uncertain.
In our study, we provide new evidence concerning the
OsABI5 gene function during plant development, espe-
cially in the regulation of plant fertility. The role of the
OsABI5 gene in the ABA signaling pathway was also
conrmed. Our results provide evidence for the involve-
ment of OsABI5 in the ABA signaling pathway and stress
tolerance. It is also possibly involved in the formation of
microspores and the regulation of plant fertility.
Acknowledgements This work was supported by grants from the
National Basic Research Program (2003CB114300), Ministry of
Science and Technology of China; National Natural Science Foun-
dation of China (30470175); and Knowledge Innovation Program,
Chinese Academy of Sciences.
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