A bZIP transcription factor, OsABI5, is involved in rice fertilityand stress tolerance

Meijuan Zou Æ Yucheng Guan Æ Haibo Ren ÆFang Zhang Æ 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) isinvolved 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 bydrought and cold (4"C) in seedlings. The OsABI5 protein

was localized in the nucleus and has trans-activation

activity. The N-terminal of the protein is necessary for itsactivity. 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 the35S promoter could rescue ABA-insensitivity of abi5-1during seed germination and result in hypersensitivity to

ABA. Over-expression of OsABI5 in rice conferred highsensitivity to salt stress. Repression of OsABI5 promoted

stress tolerance and resulted in low fertility of rice. These

results suggested that OsABI5 could regulate the adaptivestress response and plant fertility of rice as a transcription

factor.

Keywords ABA ! bZIP transcription factor ! OsABI5 !Plant fertility ! Stress endurance ! Transgenic rice

AbbreviationsABA Abscisic acid

abi3 Abscisic acid insensitive 3

abi5 Abscisic acid insensitive 5

Introduction

Throughout plant development, the phytohormone abscisicacid (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 includingseed maturation, dormancy, inhibition of cell division and

germination (Leung and Giraudat 1998). During seed

maturation in many species, ABA gradually accumulatesand triggers expression of many stress-responsive genes. In

plants, several transcription factors have been identified 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 identified by

screening for decreased sensitivity to ABA inhibition ofgermination in Arabidopsis thaliana (Finkelstein and Lynch2000). 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-responsivetranscription activators, ABI3 or its orthologs, to regulate

seed-specific and/or ABA-inducible gene expression

(Lopez-Molina et al. 2002; Nakamura et al. 2001; Hoboet 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 duringseed 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 Geneticsand Developmental Biology, Chinese Academy of Sciences,P.O. Box 2707, South 1-3, Zhongguancun, Beijing 100080,P.R. Chinae-mail: fchen@genetics.ac.cn

123

Plant Mol Biol (2008) 66:675–683

DOI 10.1007/s11103-008-9298-4

The bZIP-type transcriptional factors are involved in

developmental and physiological processes in response tostresses, 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 withspecific ABA-responsive cis-acting elements (ABRE) and

trans-activate down-stream gene expression. Using various

promoter analyses, the G-box element 50!!!CACGTG!!!30(ABRE) has been shown to be present in many stress-

responsive genes and is involved in ABA-regulated expres-sion (Niu et al. 1999;Kim et 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 toABRE in the AtEm6 promoter (Carles et al. 2002).

Rice is an important economic crop and feedsmanypeople.

Understanding themechanisms of stress responses in ricewillprovide a fundamental foundation for research on plant

endurance. At present, most reports on stress responses focus

onArabidopsis. Redundant and distinct functions of the ABAresponse loci ABI5 and ABF3 have also been found in Ara-bidopsis (Finkelstein et al. 2005). The conservation and

specificity of theABA signaling pathway and stress responsesin rice compared with Arabidopsis remains largely unknown.

In this paper, we present the isolation and identification of a

rice bZIP transcription factor, OsABI5. It displayed highhomology with the Arabidopsis AtABI5 gene, suggesting

similar functions in the adaptive stress response and the ABA

signaling pathway. We analyzed the biological function ofOsABI5 in vivo using transgenic plants. Tolerance to salt

stress and fertility of rice were affected by decreasing

expression of OsABI5. OsABI5 may have overlapping anddistinct 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 undercontrolled conditions (16 h light/8 hdark cycle) at 27"C.One-week-old seedlings were used in various stress treatments.

The Arabidopsis seedlings of wild-type Wassilewskija(Ws-2), abi5-1mutants, and transgenic plants were plated on

1/2 MS supplemented with different concentrations of ABA,

chilled for 2 days at 4"C in darkness, and incubated for 3 or12 days at 22"C with a 16 h photoperiod to compare the

germination rates.

The variety of rice used for transformation experimentswas 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 amutation 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 thathad been treated with phytohormones and/or stress, using

Trizol reagent (Gibco). Reverse transcription-PCR was

performed according to the manufacturer’s instructions(SuperScript. II RNase H- Reverse Transcriptase, Invitro-

gen). Primers 50-ATGGCATCGGAGATGAGCAAGAA

C-30 and 50-GCTTCTTTGTCAGTAGAACCGTCTTC-30

wereused to test the expressionpattern ofOsABI5 in seedlingsunder different treatments and in transgenic lines. Total RNAs

were isolated from the young panicle in rice transgenic linesto analyze gene expression. The actin gene was amplified as

an internal control to quantify the relative amounts of cDNA.

Subcellular localization of OsABI5::GFP in onion

epidermal cells

We amplified theOsABI5 cDNA with the primers containing

two attB recombination sites, subcloned the products into adonor vector by recombination in vitro (GatewayBPClonase

EnzymeMix, Invitrogen) and created an entry vector. The LR

clonase reaction to transfer DNA fragments from entry clonesto destination vectors pMDC83 was carried out according to

the manufacturer’s instructions (Invitrogen). The OsA-

BI5::GFP fusion protein driven by the CaMV 35S promoterwas introduced into onion epidermal cells with an Agrobac-terium-mediated system, incubated on 1/2 MS medium for

24 h at 26"C in darkness, and the fluorescence of GFP wasvisualized under a fluorescence microscope.

Transactivation analysis in yeast

The trans-activation assay was performed according to themethods 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 examinethe 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) 66:675–683

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analyzed by b-galactosidase assays. A colony-lift filter

assay was carried out according to the yeast handbookinstructions (Clontech).

DNA binding assay

We constructed a yeast reporter strain that carried the dualreporter genes HIS3 and lacZ, with a trimer of 27 bp DNA

fragments upstream of the TATA element composed asfollows:

50-agctAGCCACGTGTCGGACACGTGGCA-30

30-TCGGTGCACAGCCTGTGCACCGTtcga-50

The 27 bp fragment contained two G-box motifs and a

50-agct-30 annealing end, which was ligated into three

tandemly repeated copies and then inserted into the HindIIIsite 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 fromthe 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), whichhad 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 lacZminimal 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 Hisand Ura). The resulting yeast strain transcribed theHIS3 geneat 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 filter papers containing X-gal. These yeast cells

were separately transformed with the yeast expression vectorpAD-Gal4-2.1, which harbored theOsABI5 genes. The plateswere incubated for 3 days, and then analyzed by colony-lift

filter 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-AT–resistant 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 verified

by sequencing. The Arabidopsis transgenic lines (Ws-2 andabi5-1) were generated by the Agrobacterium tumefaciensfloral-dip method (Clough 1998). Seeds (T1) from infil-

trated plants were plated on 1/2 MS medium containing25 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 werecollected and stained with a 1% I2–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 highamino acid sequence homology with Arabidopsis AtABI5

and barley HvABI5 within five regions (Genetyx 5.0 soft-

ware) (Fig. 1) and was designated as OsABI5 (GenBankaccession No. EF199631). Amino acids homology analysis

showed that the OsABI5-encoded peptide contains a typical

basic leucine zipper domain. The deduced amino acidsequence 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, maturationand 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 functionaldomains 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) 66:675–683 677

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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 (4"C) initially suppressed OsABI5expression within 5 h, and then induced it to reach its

maximum at 24 h. When plants were subjected todehydration 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 theepidermal cells of onion. Protein expression was observed

under a fluorescence microscope. The result showed that

the OsABI5::GFP fusion protein was targeted to the nuclei

Fig. 1 Comparison of thededuced amino acids of OsABI5with other bZIP proteins.Multiple sequence alignment ofthe OsABI5 amino acidsequence with other bZIPprotein sequences, TRAB1(GenBank accession No.BAA83740), HvABI5(GenBank accession No.AY150676), and ABI5(GenBank accession No.AAD21438). Blast analysis wasconducted with Genetyxsoftware (5.0). The identicalresidues are shaded in black.The basic domain and leucinerepeats of the bZIP domain aredouble-underlined andconserved regions are single-underlined. Asterisks (*) denotethe 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 ABAand stress conditions. Total RNAs were isolated from 7-day-old riceseedlings treated with 100 lM ABA, cold (4"C), drought, and250 mM NaCl at the indicated times. The length of PCR products is1140 bp. The rice actin gene was used as an internal control

Fig. 3 Nuclear localization of the OsABI5 protein. OsABI5::GFPfusion protein was expressed transiently under the control of CaMV35S promoter in onion epidermal cells and observed under afluorescence microscope. The photographs were taken in dark fieldfor green fluorescence (b), in bright light for the morphology of thecell (a), and in combination (c)

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of the cells (Fig. 3). These data suggested that OsABI5 was

a nuclear protein and functioned as a transcription factor toregulate 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 entirecoding region and several N-terminal regions of OsABI5with 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 yeastcells 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 notgrow on selection medium (Fig. 4d). A colony-lift filter

assay supported these results (Fig. 4e). These results showed

that the full-lengthOsABI5 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 portioncontaining the bZIP domain. This indicated the necessity of

the N-terminal region for transactivation activity.

The bZIP protein is known to interact mainly with DNAcis-elements containing a 50-ACGT-30 core sequence. The

binding specificity 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-mediatedsystem. The positive transgenic lines were confirmed by

PCR amplification using primers from hygromycin (data

not shown). In total, 13 independent antisense transgenicplants 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%. Theantisense 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 degreesand the decreased fertility rate of rice was correlated with

the expression of OsABI5 (Fig. 5a).

Microarray data showed that the OsABI5 gene was highlyexpressed 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 transformedplants to investigate the pollen status. The pollen grains of the

control were stained uniformly using I2-KI staining. How-

ever, in transgenic lines more than 60% of microspores inmature pollen showed an abnormal shape and did not stain,

which suggests they are sterile (Fig. 5c). Therefore we

speculated that suppression of theOsABI5mRNA levelmightcause 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 activityof the OsABI5 protein. Fusionproteins of the GAL4 DNAbinding domain and differentportions of OsABI5 wereexpressed in yeast strain Y187.The transformed yeast culturewas dropped onto YPDA or SDplates without histidine andgrowth status examined. Theplates were incubated for3 days, and then analyzed byb-galactosidase assay. pGAL4and pBD were used as positiveand negative controls,respectively

Plant Mol Biol (2008) 66:675–683 679

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Stress tolerance of OsABI5 transgenic plants

To examine the in vivo function of OsABI5 in stress

responses, we used plants over-expressing OsABI5, andOsABI5-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 thetolerance of OsABI5 over-expression plants and OsABI5-suppressed plants to high salt concentration or PEG

treatment. The antisense OsABI5 transgenic plants grewwell in high salt and exhibited higher tolerance to salt,

which indicated that the suppression of OsABI5 expres-

sion increased salt tolerance. In contrast, higherconcentrations 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 theexpression 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 stresstolerance of rice OsABI5 transgenic lines. 35S::OsABI5(+) and35S::OsABI5(-) denote the OsABI5-over-expression and antisensetransgenic lines, respectively. (a) RT-PCR analysis of antisensetransgenic lines L2, L3, L6 and their fertility rates. RT-PCR wasperformed for 37 cycles to investigate the OsABI5 expression. Therice actin gene was used as an internal control. Plants carrying anempty vector were controls. (b) Comparison of rice fertility status incontrol and antisense transgenic lines. (c) Microspore status of mature

pollen stained by K–I2 (d) Stress tolerance of transgenic lines underNaCl treatment. 20-day-old seedlings of rice transgenic lines wereexposed to 250 mM NaCl for 25 days. (e) Stress tolerance oftransgenic lines under PEG treatment. Seedlings (20-days-old) of thecontrol and antisense transgenic lines were exposed to 15% PEG for5 days. (f) RT-PCR analysis of OsABI5-over-expression transgeniclines. Lanes 1 and 2: rice over-expression transgenic lines. Plantscarrying an empty vector were controls. RT-PCR was performed for30 cycles to investigate OsABI5 expression

680 Plant Mol Biol (2008) 66:675–683

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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 (Rabbaniet 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 intransgenic 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 theantisense transgenic lines and vector controls at the panicle

stage. RT-PCR results showed that respressing OsABI5expression altered the expression of aba45, aba2, SalT,SKC1 andWsi18. No change was found in the expression ofOsLea3, Lip9, OsEMP1 (Fig. 6). Interestingly, the Wsi18

gene has two transcripts (unpublished data) and their

expression was altered differently. The expression of SKC1was 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 couplingbetween stress tolerance andOsABI5 function. These resultssuggest the existence of a complex regulatory relationship

between the transcription factor OsABI5 and the expressionof other genes.

Discussion

bZIP transcription factors play important roles in diverse

biological processes such as stress signaling, seed matu-

ration and flower development (reviewed by Finkelsteinet al. (2002)). In the present study, we reported the isola-

tion and characterization of a rice gene, OsABI5, whichbelongs 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 inthe stress response and ABA signal transduction.

The OsABI5 protein was constitutively localized to the

nucleus. Other bZIP proteins of the ABI5 subfamily arealso 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 theOsABI5 protein functions as a transcriptional activator in

yeast (Fig. 4). Deletion analysis showed that the N-termi-

nal region is necessary for transcriptional activation. Thesedata 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 atranscription factor to regulate the down-stream genes.

The expression of OsABI5 was induced by ABA and

stress treatment and may have an important physiologicalfunction 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 findings suggest thatOsABI5 functions as a transcriptional activator in ABA

signal transduction. A complementation test showed that

OsABI5 could rescue the ABA sensitivity of abi5-1, andtransgenic 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-responsivegene expression in the different OsABI5 antisense transgenic lines.The rice actin gene was used as an internal control. L2, L3, L6denoted the different antisense lines. Plants carrying an empty vectorwere controls

Plant Mol Biol (2008) 66:675–683 681

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WT during seed germination. Meanwhile, over-expression

of OsABI5 in WT Ws-2 resulted in the WT displayinghypersensitivity 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 playsa role in protecting germinating embryos from drought, and

ABI5 is regulated by ABA during germination (Lopez-

Molina et al. 2001). Based on the complementationexperiments and the similar phenotype of over-expression

transgenic lines, we propose that OsABI5 has a similarfunction 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 inopposite phenotypes in stress responses (Fig. 5d). The

seedlings of the OsABI5 over-expressed plants were sen-

sitive to salt stress, while the OsABI5-repressed plantsshowed increased stress resistance to NaCl and PEG. This

result suggests that stress-responsive signaling is activated

in the OsABI5-repressed plants. The results show thatOsABI5 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 atight 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 expressionof 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 alsoindicated that OsABI5 expression might be an important

mediator of these genes’ expression, and that OsABI5 is an

important determinant of ABA and salt signaling. Theexpression 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 theantisense 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 maymodulate formation of mature pollen. ABA promotes

acquisition of desiccation tolerance during seed maturation

(Ooms Jaap et al. 1994). In Arabidopsis, the functionof AtABI5 genes in pollen development is unidentified.

AtABI5 is expressed in vegetative and floral 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

confirmed. 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 theNational Basic Research Program (2003CB114300), Ministry ofScience and Technology of China; National Natural Science Foun-dation of China (30470175); and Knowledge Innovation Program,Chinese Academy of Sciences.

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