Plant Mol Biol (2012) 80:571–585

DOI 10.1007/s11103-012-9967-1

Function of the HD-Zip I gene Oshox22 in ABA-mediated drought

and salt tolerances in rice
Shuxin Zhang • Imran Haider • Wouter Kohlen • Li Jiang •
Harro Bouwmeester • Annemarie H. Meijer • Henriette Schluepmann •

Chun-Ming Liu • Pieter B. F. Ouwerkerk

Received: 24 February 2012 / Accepted: 4 September 2012 / Published online: 30 October 2012
Ó Springer Science+Business Media B.V. 2012

Abstract Oshox22 belongs to the homeodomain-leucine ABA content, decreased sensitivity to ABA, and enhanced
zipper (HD-Zip) family I of transcription factors, most of which tolerance to drought and salt stresses at the seedling stage. In
have unknown functions. Here we show that the expression of contrast, transgenic rice over-expressing Oshox22 showed
Oshox22 is strongly induced by salt stress, abscisic acid (ABA), increased sensitivity to ABA, increased ABA content, and
and polyethylene glycol treatment (PEG), and weakly by cold decreased drought and salt tolerances. Based on these results,
stress. Trans-activation assays in yeast and transient expression we conclude that Oshox22 affects ABA biosynthesis and reg-
analyses in rice protoplasts demonstrated that Oshox22 is able ulates drought and salt responses through ABA-mediated signal
to bind the CAAT(G/C)ATTG element and acts as a tran- transduction pathways.
scriptional activator that requires both the HD and Zip domains.
Rice plants homozygous for a T-DNA insertion in the promoter Keywords Rice  Transcription factor  HD-Zip 
region of Oshox22 showed reduced Oshox22 expression and Drought stress  Regulation  Abiotic stress

Abbreviations
Electronic supplementary material The online version of this HD-Zip Homeodomain-leucine zipper
article (doi:10.1007/s11103-012-9967-1) contains supplementary
material, which is available to authorized users.
ABA Abscisic acid
PEG Polyethylene glycol
S. Zhang  L. Jiang  C.-M. Liu (&)  P. B. F. Ouwerkerk (&) GFP Green fluorescent protein
Key Laboratory of Plant Molecular Physiology, Institute RT Reverse transcription
of Botany, Chinese Academy of Sciences,
PCR Polymerase chain reaction
Beijing 100093, China
e-mail: p.b.f.ouwerkerk.2@gmail.com HB Homeobox
C.-M. Liu
e-mail: cmliu@ibcas.ac.cn
Introduction
S. Zhang  L. Jiang
Graduate School of Chinese Academy of Sciences, Beijing Drought and salt are major abiotic stresses that cause tre-
100049, China
mendous yield losses in crops all over the world. Due to
S. Zhang  A. H. Meijer  P. B. F. Ouwerkerk water shortage and less predictable rainfall patterns
Institute of Biology, Leiden University, P.O. BOX 9505, 2300 resulting from global atmospheric changes, the improve-
RA Leiden, The Netherlands ment of stress resistance in crops is now of utmost
I. Haider  W. Kohlen  H. Bouwmeester importance. Consequently, the genetic basis of drought and
Laboratory of Plant Physiology, Wageningen University, salt resistance is an intensively studied topic. The plant
Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands hormone abscisic acid (ABA) plays a central role in
drought and salt responses through regulating develop-
H. Schluepmann
Department of Molecular Plant Physiology, Utrecht University, mental and physiological processes including stomata
Padualaan 8, 3584 CH Utrecht, The Netherlands closure (Leung and Giraudat 1998; Umezawa et al. 2009;

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Melcher et al. 2010; Huang et al. 2012). Using microarrays, Oshox22 regulates drought and salt stress susceptibility
many genes with expression responding to ABA, drought through an ABA-mediated signaling pathway.
and salt treatments have been identified (Seki et al. 2001,
2002; Rabbani et al. 2003; Bray 2004; Yamaguchi-Shino-
zaki and Shinozaki 2006). Additional characterization Materials and methods
identified key components mediating gene-expression
changes in drought responses that include the transcription Plant materials and stress treatments
factors DREB1A (Kasuga et al. 2004), DREB2A (Sakuma
et al. 2006), SNAC1 (Hu et al. 2006), OsbZIP23 (Xiang Drought-tolerant cultivar IRAT 112 (upland tropical
et al. 2008), and DST (Huang et al. 2009). Furthermore, a japonica, also named Gajah Mungkur) and drought-sensi-
number of transcription factors encoded by homeodomain- tive cultivar Nipponbare (lowland japonica) were used for
leucine zipper (HD-Zip) genes in Arabidopsis, rice and most studies described in this paper. Zhonghua 11 (lowland
other plants have been implicated in regulating drought japonica) was used for transgenic analyses. The T-DNA
tolerance through either ABA-dependent or ABA-inde- insertion mutant oshox22-1 in Dongjin (lowland japonica)
pendent pathways (e.g. Söderman et al. 1996, 1999; Gago background was obtained from the Postech collection in
et al. 2002; Himmelbach et al. 2002; Deng et al. 2006; South Korea.
Agalou et al. 2008; Shan et al. 2011). The HD-Zip genes, For hormone treatments, 12 day-old Nipponbare seed-
however, are an abundant group of transcription factors lings were sprayed with 100 lM ABA, followed by sam-
that are exclusively found in plants (Ruberti et al. 1991; pling at 0, 1, 3, and 6 h. Alternatively, seedlings at the
Schena and Davis 1992; Aso et al. 1999; Sakakibara et al. same stage were irrigated with 10 % PEG 6,000 or
2001; Derelle et al. 2007). HD-Zip proteins, characterized 200 mM NaCl followed by sampling at 0, 1, 3, and 6 h. For
by a DNA-binding HD and a protein–protein interaction the cold treatment, seedlings were transferred to 4 °C and
Zip domain, have been classified into four families (I–IV) sampled after 0, 1, 3, 6, 12, and 24 h. One whole plant was
according to their sequence similarities (Ruberti et al. sampled as one replicate and in total four replicates were
1991; Morelli and Ruberti 2002). Different members of the used in each RNA extraction.
HD-Zip families I and II have been implicated in auxin For drought treatments, about 40 plants of the oshox22-1
signaling and transport (Morelli and Ruberti 2002; Sawa mutant and Oshox22 over-expression lines were grown in
et al. 2002), vascular development (Scarpella et al. 2000), square pots (L26 9 W12 9 H10 cm) filled with a mixture
and light responses including shade avoidance (Steindler of sand and soil (1:1) together with wild type, respectively.
et al. 1999; Wang et al. 2003). Several other members, such We stopped watering when seedlings were 12 days old,
as Athb-6, Athb-7 and Athb-12 from Arabidopsis and watering was resumed for 3 days when seedlings were
(Söderman et al. 1996, 1999; Lee et al. 2001; Hjellström 24 days old, after which the survival rates were calculated.
et al. 2003; Olsson et al. 2004), Hahb-4 from sunflower For salt treatments, the same amount of 12 day-old seed-
(Gago et al. 2002; Dezar et al. 2005a, b; Manavella et al. lings were irrigated with 150 mM NaCl for 9 days, after
2006), and CpHB2, CpHB6 and CpHB7 from Cratero- which green leaf rates (green leaf area[30 %) and survival
stigma plantagineum (Frank et al. 1998; Deng et al. 2002) rates were determined. To measure the water-loss rate
are induced by drought and ABA, suggesting a function in under dehydration conditions, the second leaves from the
ABA-mediated adaptation to drought stress. In agreement, top of the plant at the tillering stage (plants were 55 days
inductions of Athb-6, Athb-7 and Athb-12 are abolished in old) were cut and exposed to air at room temperature
the ABA-insensitive mutants abi1 and abi2 (Himmelbach (approximately 25 °C), and the weight was determined
et al. 2002; Olsson et al. 2004). every 30 min. Every treatment was done in triplicate.
Like in dicots, a subset of HD-Zip family I and II genes is
regulated by drought in rice (Agalou et al. 2008). Phyloge- Subcellular localization assay
netic analysis places Oshox22, Oshox24, Athb-7 and Athb-12
in the same subgroup (c-clade, Henriksson et al. 2005) of the A full-length cDNA of Oshox22 was amplified from IRAT
HD-Zip family I, and Oshox22 is very likely related to 112 by RT-PCR using primers Oshox22cdsFW and Osh-
Oshox24 via an ancient chromosomal duplication (Agalou ox22cdsRW (Table S1) based on a full-length cDNA
et al. 2008). Our previous work showed that Oshox22 is sequence (GenBank accession AY224440) found in a seed-
strongly induced by drought which spurred our interest for derived cDNA expression library (Cooper et al. 2003). The
further studies (Agalou et al. 2008). To gain insight into the PCR product was cloned into pCR2.1-TOPO (Invitrogen)
function of Oshox22 in drought and salt tolerances, we per- and sequenced. To create a GFP-tagged construct, the full-
formed genetic and physiological studies through mutation length Oshox22 cDNA was excised by EcoRI and ligated
and over-expression analyses in rice. Our data showed that into EcoRI-digested pTH2-BN vector. The cDNA fragment

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was inserted to the C-terminus of GFP and expressed using Rice protoplast isolation and transient expression
the Cauliflower mosaic virus (CaMV) 35S promoter. The assays
construct was transformed into rice protoplasts as previ-
ously described (Chen et al. 2006; Osnato et al. 2010). Assays to test DNA binding of Oshox22 involved transient
Localization of the GFP-tagged Oshox22 protein was transformations of protoplasts with effector and reporter
monitored in protoplasts by confocal laser scanning plasmids. The effector plasmid contained the full-length
microscopy (Leica SP5) at 24 h after transformation. cDNA of Oshox22 expressed under the control of the
CaMV 35S promoter. The reporter plasmids contained the
Yeast one-hybrid screens putative Oshox22 binding sequences AH1 or AH2 as tet-
ramers upstream of a truncated -90 CaMV 35S promoter
To assay the activation property, the full-length Oshox22 directing GUS gene expression (Meijer et al. 1997). For
cDNA PCR product was excised by EcoRI and BamHI, protoplast isolation, one hundred rice seeds (Nipponbare)
then ligated into EcoRI/BamHI digested pAS2-1 (Clontech, were grown in 10 cm diameter pots (28 °C, 85 % humid-
GenBank accession U30497), resulting in pAS2-1- ity) in the dark for 12–14 days. Stems and leaves were cut
Oshox22. Partial Oshox22 ORFs were also cloned into into *0.5 mm pieces and digested with 25 ml enzyme
pAS2-1 vector, producing different translational fusions solution containing 1.5 % w/v cellulase (Sigma) and 0.3 %
between Oshox22 and the GAL4 BD. Constructs were w/v Macerozyme (Sigma) in 50 ml centrifuge tubes. Fur-
sequence-verified before transfer to yeast strain PJ69-4A ther preparation of protoplasts and transfection with
(Table S2) for activation screens. All yeast handling and effector/reporter constructs were as described earlier (Chen
reporter assays were performed as described before (Meijer et al. 2006; Osnato et al. 2010). Proteins extraction and
et al. 1998, 2000a; Ouwerkerk and Meijer 2001, 2011). detection of GUS activity were based on Jefferson et al.
For the DNA binding assay, a full-length cDNA of (1987). Fluorescence was measured by a Cytofluor 2350
Oshox22 was excised from pAS2-1-Oshox22 with EcoRI fluorimeter (Millipore).
and SalI and then ligated into pRED-ATGa which is rep-
licated via ARS-CEN and maintained via URA3 selection Northern blot hybridization
(unpublished results, P.B.F. Ouwerkerk and A.H. Meijer).
The resulting plasmid pRED-ATG-Oshox22 was assayed in Electrophoresis and northern blotting of RNAs were per-
yeast strains YM4271-4AH1-HIS3 and YM4271-4AH2- formed as described by Memelink et al. (1994). Baked
HIS3 which were made using pINT1 as the integrative blots were pre-hybridized in 1 M NaCl, 1 % SDS, 10 %
vector system (Meijer et al. 1998) which contains the HIS3 dextrane sulphate and 50 lg/ml denatured herring sperm
reporter gene preceded by AH1 or AH2 tetramer-binding DNA at 65 °C, washed with 0.1XSSPE, 0.5 % SDS at
sites for HD-Zip proteins (Meijer et al. 2000b). 42 °C and then autoradiographed. Probes were labeled by
random priming with 32P-dCTP. Equal loading of RNA
Binary vector constructions and plant transformation samples was verified on the basis of ethidium bromide
staining of ribosomal RNA bands.
The Oshox22 over-expression construct was made in the
binary vector pC1300intB-35SnosEX (Genbank accession Real-time qPCR analysis
AY560325) as following: the full-length cDNA of
Oshox22 was excised from pAS2-1-Oshox22 and ligated Total RNAs from different tissues were pre-treated with
into binary vector pC1300intB-35SnosEX, allowing the RNase-free DNase I (Takara), according to the manufac-
gene to be expressed under the control of the CaMV 35S turer’s instruction. Reverse transcription reaction was
promoter. We transformed rice (Zhonghua 11) as previ- performed with SuperScriptTM III reverse transcriptase
ously described (Scarpella et al. 2000) except that the (Invitrogen) following the manufacturer’s instruction.
Agrobacterium tumefaciens strain used was LBA4404. Primers used for real-time PCR analyses of Oshox22 were
Calli used for transformation were obtained from germi- QPCR-22FW and QPCR-22RW (Table S1). qPCR was
nating seeds according to Rueb et al. (1994). Plantlets were performed with Rotor-Gene 3000 Real-time PCR System
maintained in culture on half-strength Murashige Skoog using SYBR1 Green to monitor dsDNA synthesis. Relative
(MS) medium with 10 g/l sucrose until transfer to a expression levels of reporter and target genes were deter-
greenhouse (28 °C under a 16 h photoperiod and 85 % mined by the Two Standard Curves Relative Quantification
humidity). Method using ACTIN1 (Table S1) as the internal control.

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Determination of ABA content Expression of Oshox22 under abiotic stress conditions

Five seeds each of the homozygous T-DNA insertion First, RNA samples from different rice tissues at several
mutant oshox22-1 and over-expression lines together with developmental stages were analysed by quantitative RT-PCR
their corresponding wild type cultivars (Dongjin and (qRT-PCR) in Nipponbare. The results showed that Oshox22
Zhonghua 11 respectively) were surface-sterilized and is ubiquitously expressed, with a lower level in stems and
germinated in half-strength MS salt solution without higher in panicles and seeds (Fig. 1a), which is consistent with
sucrose and grown at 28 °C with a 16/8 h light/dark pho- earlier observations (Agalou et al. 2008). We then monitored
toperiod. To determine the ABA level in response to the Oshox22 expression under different abiotic stress condi-
drought stress, 12 day-old oshox22-1 and over-expression tions. As shown in Fig. 1b, Oshox22 was rapidly and strongly
seedlings were stopped watering and sampled after 10 days induced by NaCl, PEG and ABA, and weakly induced by low
dehydration treatment. Quantification of ABA levels was temperature. These results are in agreement with available
performed using LC–MS/MS with five biological replicates microarray data (http://red.dna.affrc.go.jp/RED/).
(each 0.2 g of fresh shoot tissue) as described elsewhere,
(López-Ráez et al. 2008, 2010). Statistical analyses were Subcellular localization and transcriptional activation
performed using Student’s t test. function of Oshox22

To confirm that Oshox22 is a nuclear protein, a GFP-tag-

Stomata density test ged Oshox22 construct under the control of the CaMV 35S
promoter was made, with GFP fused at the C-terminus of
Stomata numbers were counted on the upper epidermis of the full-length Oshox22 protein (construct 35S::GFP-
the leaf blades of mutant and over-expression lines grown Oshox22). This construct was introduced into rice protop-
under normal conditions. Leaf blades were from flag leaves lasts by transient transformation and the fusion protein’s
at the stage that the plants just started to flower. For this subcellular localization was analysed using confocal laser
method, oval surfaces spanning 1 9 2 cm were painted scanning microscopy, where the 35S::GFP construct
with clear fingernail polish, while avoiding ribbed veins. served as control. As shown in Fig. 1c, the GFP signal was
After the polish had dried it was peeled off by adding a detected specifically in the nuclei of 35S::GFP-Oshox22
tape on the polish and transferred to a microscopy slide. transformed protoplasts, while in the control the GFP sig-
Finally, the stomata number was counted using DIC nal was located primarily in the cytoplasm. These data
microscopy. Five replicates of every leaf were counted and suggest that Oshox22 is a nuclear-localized protein. In
from every plant line seven plants were used and all agreement with this observation, a putative nuclear local-
numbers were converted to number of stomata per square ization signal sequence (RKRR at the AA 59) was found
mm to account for variation in microscopes. near its N-terminus, as analyzed by WoLF PSORT
(http://wolfpsort.seq.cbrc.jp/).
To test whether Oshox22 has any transcription activa-
Results tion property, Oshox22 was fused to the Gal4p binding
domain (GAL4 BD) and assayed for the ability to induce
Isolation and sequence analysis of Oshox22 expression of a HIS3 gene preceded by Gal4p binding sites.
The result showed that the transformed yeast cells were
Previous studies showed that the expression of Oshox22 in able to grow on medium lacking histidine, with up to
rice is strongly induced by drought, and that the induction 10 mM 3-amino-1,2,4-triazole (3-AT), a competitive
is higher in three drought tolerant upland cultivars com- inhibitor of His3p enzyme activity (Fig. 2a). In contrast, no
pared with three lowland cultivars (Agalou et al. 2008). To growth was observed of yeast transformed with a control
elucidate the function of Oshox22, the full-length cDNA of construct without Oshox22 (pAS2-1), indicating that
Oshox22 was amplified from the rice cultivar IRAT 112 by Oshox22 has transcriptional activation activity in yeast. To
RT-PCR. The sequence of the amplified cDNA fragment dissect the activation domain(s), a series of seven truncated
was identical to that of the cDNA from Nipponbare over Oshox22 constructs with deletions from either the N- and
the whole length. Oshox22 (Os04g45810) is located on C-termini were tested. No activation activity was observed
chromosome 4 and encodes a protein of 262 amino acids when either the HD or the Zip domains were deleted
(AA) including a 61-AA HD domain for DNA binding and (Fig. 2a), suggesting both domains are required for tran-
a 43-AA Zip domain for protein–protein interactions. scriptional activation in yeast.

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Fig. 1 Expression profiling and

subcellular localization of
Oshox22. a qRT-PCR analyses
of Oshox22 expression in
scutellum-derived calli, root,
shoot, leaf sheath, flag leave,
panicle, stem and mature grain
from rice. b Northern blot
analysis of Oshox22 expressions
in response to ABA (100 lM),
salt (200 mM NaCl), 10 % PEG
6,000 and low-temperature
(4 °C) treatments. c Nuclear
localization of GFP-tagged
Oshox22. The GFP-Oshox22
fusion protein, driven by the
CaMV 35S promoter, was
transiently expressed in rice
protoplasts and visualized by
confocal laser scanning
microscopy. Construct pTH2
(Chiu et al. 1996) carrying a
GFP gene driven by CaMV 35S
promoter was used as the
negative control

Interactions of Oshox22 in yeast and rice chromosomally integrated HIS3 reporter gene with
with the CAAT(C/G)ATTG sequence upstream AH1 or AH2 tetramers (named 4AH1-HIS3 and
4AH2-HIS3 respectively, Meijer et al. 1998, 2000b) were
Previous results showed that HD-Zip family I and II pro- used. The Oshox22 ORF was cloned into pRED-ATGa
teins are able to interact with pseudopalindromic AH1 (named pRED-ATGa-Oshox22), allowing for constitutive
(CAAT(A/T)ATTG) and AH2 (CAAT(C/G)ATTG) expression of full length Oshox22 protein in yeast without
sequences, respectively (Sessa et al. 1993; Meijer et al. fusion to an exogenous activation domain. Construct
1997, 1998, 2000b; Palena et al. 1999; Johannesson et al. pRED-ATGa-Oshox22 was transformed into yeast strains
2001). To test whether Oshox22 interacts with either the containing constructs 4AH1-HIS3 or 4AH2-HIS3. The
AH1 or AH2 sequences or both, yeast strains containing a results showed that yeast cells with 4AH2-HIS3

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Fig. 2 Trans-activation and

DNA-binding specificity of
Oshox22. a Trans-activation
assay in yeast with truncated
Oshox22 protein. Fusion
proteins of the GAL4 DNA-
binding domain and different
fragments of Oshox22 were
examined for their trans-
activation activities in yeast
PJ69-4A. HD, homeodomain;
Zip, leucine zipper. b Trans-
activation assay of Oshox22 in
yeast. Sections 1 and 2, negative
control; sections 3 and 4,
Oshox22 fused to GAL4-BD in
the vector pRED-ATGa was
transformed into yeast strains
YM4271-4AH1-HIS3 and
YM4271-4AH2-HIS3,
respectively. c Interactions of
Oshox22 with the HD-Zip
binding site AH2 (CAAT(C/
G)ATTG) and activation of
reporter gene expression in a
transient expression system
using rice protoplasts. Transient
expression of Oshox22 was
driven by the CaMV 35S
promoter. The Oshox22-OX
construct was co-transformed
with the reporter constructs
GUSXX-4AH1 or GUSXX-
4AH2. The empty vectors
pRT101 and GUSXX-90 were
used as negative controls

transformed with pRED-ATGa-Oshox22 grew well on a To confirm binding of Oshox22 protein to the AH2
medium lacking histidine but containing up to 10 mM sequence transient expression assays were carried out with
3-AT (Fig. 2b), whereas no growth was observed in yeast effector and reporter plasmids in rice protoplasts. Two
strains with 4AH1-HIS3 or with empty pRED-ATGa vector. reporter plasmids, 4AH1-90-GUS and 4AH2-90-GUS were
Thus, in yeast, Oshox22 is able to bind AH2, but not AH1, used, in which the AH1 and AH2 tetramers were fused to a
and can activate reporter gene expression by an intrinsic CaMV -90 35S minimal promoter. Construct Pro35S-
activation domain. Oshox22 with Oshox22 expressed under control of the

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Fig. 3 The phenotype and

ABA sensitivity of oshox22-1.
a Morphology of oshox22-1 at
post-anthesis stage. b Schematic
representation of the exon–
intron structure of Oshox22
gene and the position of the
T-DNA insert in oshox22-1.
c Oshox22 expression was
down-regulated in oshox22-1.
d Germination rate of oshox22-
1 on MS medium with 0, 3, 5,
and 8 lM ABA for 3 days.
e Relative growth of oshox22-1
on medium with 5 lM ABA at
the post-germination stage.
Dongjin represent wild type
plants segregated from a
heterozygous oshox22-1 parent

CaMV 35S promoter was used as an effector. As shown in oshox22-1 (Fig. S1). Northern blot analysis showed that in
Fig. 2c, GUS expression in protoplasts co-transformed with oshox22-1 the Oshox22 transcript was below detection
Pro35S-Oshox22 and 4AH2-90-GUS was 3.07 times higher level (Fig. 3c). Phenotypic studies showed that oshox22-1
than those co-transformed with Pro35S-Oshox22 and plants exhibited no obvious morphological difference
4AH1-90-GUS, and 6.11 times higher than those co- compared to wild type Dongjin (Fig. 3a). The panicle
transformed with the empty vector. These data indicate that shape and the grain number remained unchanged. When
Oshox22 is capable to activate transcription of the reporter oshox22-1 was backcrossed with Dongjin, all F1 and F2
gene when upstream HD-Zip binding sites AH1 or AH2 are plants showed the same plant stature in the greenhouse. In
present (Fig. 2c). The interaction is less effective at the the F2 population, the T-DNA insert segregated in a 3:1
AH1 site than that at AH2, which is in contrast to earlier ratio (n = 326), suggesting no embryo or gamete lethality
observations where HD-Zip I proteins mainly interacted was involved.
with AH1 and HD-Zip II proteins with AH2 (Sessa et al. We further analysed the sensitivity of oshox22-1 to
1993; Meijer et al. 1997, 2000b; Palena et al. 1999). ABA in germination assays. On MS media with 3 and
8 lM ABA, seeds from oshox22-1 showed significantly
Decreased ABA sensitivity of the oshox22-1 mutant higher germination rates (72 and 32 %, respectively) than
those from wild type plants segregated from oshox22-1
We searched the publicly available mutant collections and backcrosses (40 and 8 % on the medium with 3 and 8 lM
obtained a putative T-DNA insertion mutant for Oshox22 ABA, respectively; Fig. 3d). On control media without
in rice (POSTECH_C054121, B-11507) (Jeong et al. 2006) ABA, the germination rates of oshox22-1 and wild type
(Fig. 3a). The genomic locus of Oshox22 in this mutant is seeds showed no significant difference. These results sug-
shown schematically in Fig. 3b. Alignment of the flanking gest that Oshox22 may mediate ABA sensitivity. Next,
sequence tag and the genomic sequence showed that the oshox22-1 seedlings were tested for ABA sensitivity at the
T-DNA insertion is located at 978 bp before the transla- post-germination stage using media with different
tional start codon of Oshox22. To investigate the function concentrations of ABA. The results showed that shoot
of Oshox22, an homozygous T-DNA insertion line (named development was less inhibited by exogenous ABA in
oshox22-1) was identified, and Southern blot analysis oshox22-1, as compared to wild type (Fig. 3e). In sum-
showed that only one copy of T-DNA was present in mary, down-regulation of Oshox22 expression in the

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Fig. 4 Phenotype and ABA

sensitivity of Oshox22-OX
plants. a Phenotype of two
independent Oshox22-OX lines
at mature stage. b Germination
rate of seeds from Oshox22-OX
plants on medium with 0, 3, 5,
and 8 lM ABA for 3 days.
c Shoot and root growth of
Oshox22-OX plants on MS
medium with 5 lM ABA at
post-germination stage. ZH11
(Zhonghua 11), wild type
segregated from the T1 line

T-DNA insertion line led to compromised ABA sensitivity germination rates observed in Oshox22-OX seeds could be
at germination as well as post-germination stages. due to an increased ABA sensitivity. We further tested
Oshox22-OX seedlings on media containing 0, 3, 5 or
Increased ABA sensitivity of transgenic plants over- 8 lM ABA and found that the growth of Oshox22-OX
expressing Oshox22 seedlings was inhibited to a greater extent by 5 lM ABA
(Fig. 4c). We therefore concluded that over-expression of
For over-expression analysis we expressed Oshox22 under Oshox22 led to increased ABA sensitivity at the germina-
the control of the CaMV 35S promoter (construct tion as well as post-germination stages.
Oshox22-OX). Northern blot analysis confirmed that
Oshox22 gene expression levels were increased in the Functions of Oshox22 in regulating ABA biosynthesis
transgenics (Fig. S2). Compared to wild type control
plants, the Oshox22-OX plants exhibited fewer tillers and Next, endogenous ABA levels were measured of the
decreased height (Fig. 4a). We chose two transgenic lines Oshox22 mis-expression plants grown under control and
(Oshox22-OX-2 and Oshox22-OX-17) for further analyses. drought-stress conditions (Fig. 5). Under normal irrigated
The seed germination rates were reduced 17 % in these two conditions, the ABA content of wild type Dongjin seed-
lines (Fig. 4b). To analyse the sensitivity of the Oshox22- lings was 9.92 ± 1.06 ng g-1 FW, while in oshox22-1 it
OX plants to ABA, seeds were germinated on solid MS was reduced to 3.86 ± 0.86 ng g-1 FW, an average
media containing either 0, 3, 5 or 8 lM ABA. As shown in reduction of 60 % (Fig. 5a). In wild type Zhonghua 11, the
Fig. 4b, germination of seeds from both Oshox22-OX lines ABA content was 6.38 ± 0.72 ng g-1 FW, while in over-
was severely inhibited by all concentrations of ABA used expression lines Oshox22-OX-2 and Oshox22-OX-17, the
and the inhibition was much stronger than that observed for ABA contents were increased to 7.69 ± 0.42 ng g-1 FW
wild type seeds. For instance, the germination rates of the and 8.35 ± 0.78 ng g-1 FW, respectively (Fig. 5a). Thus,
two Oshox22-OX lines plated on medium with 3 lM ABA down-regulation of Oshox22 led to a reduced level of ABA
were 40 %, as compared to 80 % on medium without and over-expression led to increased ABA levels, therefore
ABA, whereas in the wild type only a slight reduction we conclude that Oshox22 functions in regulating biosyn-
(from 98 to 80 %) was observed. The relatively low thesis or degradation of ABA in rice. The differences in

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When the seedlings were 12 days old (five leaf stage) we

stopped watering for 12 days and then resumed watering
for 3 days to measure the survival rates. Drought treatment
strongly decreased the survival rate of Oshox22-OX plants,
while in oshox22-1 mutants the survival rate was increased,
as compared to their corresponding wild type control
groups (Fig. 6a, b). As shown in Fig. 6c, d, 92 % of the
oshox22-1 seedlings survived, as compared to 34.7 % in
the control group. In contrast, 63 % of the Oshox22-OX
seedlings survived, as compared to 87 % in their control
group. From these data we conclude that mutation of
Oshox22 led to increased drought tolerance, while over-
expression of Oshox22 led to decreased drought tolerance.
Furthermore, we measured water-loss rates in leaves from
oshox22-1 and Oshox22-OX plants during the dehydration
process. Leaves from oshox22-1 had significantly lower rates
(P \ 0.05) of water-loss than control plants (Fig. 6e). In
contrast, Oshox22-OX plants showed no significant differ-
ence in water-loss rate as compared to the control (Fig. 6f).
Because stomata density is a critical factor in drought stress,
we determined the stomata density in leaf blades of oshox22-
1 plants at flowering stage, Oshox22-OX, and their corre-
sponding wild type control plants. The oshox22-1 leaves
showed 17.8 % reduction in stomata density, but stomata
Fig. 5 Effects of Oshox22 mis-expression on ABA levels. ABA density remained unchanged in Oshox22-OX, as compared
levels in oshox22-1 and Oshox22-OX plants and wild type cultivars to leaves from wild type plants (Fig. 7). Therefore, the
Dongjin and Zhonghua 11. a ABA levels under normal condition. enhanced drought tolerance in oshox22-1 may partly result
b ABA levels after drought treatment. After drought treatment, the
from decreased stomata density in leaves.
ABA levels increased with a factor 21.39, 38.10, 9.34, 15.04 and
15.04 for oshox22-1, Dongjin, Zhonghua 11, Oshox22-OX-2 and To evaluate salt tolerance in oshox22-1 and Oshox22-
Oshox22-OX-17, respectively OX plants, 12-day-old seedlings grown in hydroponic
culture were transferred to a 150 mM NaCl solution for
9 days and then the green leaf area was measured. The
ABA levels are likely causing changes in sensitivity of oshox22-1 plants had significantly more green leaf area
germinating and developing seedlings towards exogenous (80 %) than control plants (36 %; Fig. 8). In contrast,
applied ABA. Furthermore, we also tested the ABA con- Oshox22-OX plants had a reduced green leaf area (65 %)
tents under drought stress conditions. We found that ABA compared with the control (86 %; Fig. 8). These results
contents were increased in all plants after drought treat- suggest that in rice, down-regulation of Oshox22 improved
ment (Fig. 5b), however, there were differences in the salt tolerance, whilst over-expression of Oshox22 led to
levels of induction ranging from 9.34 in wild type reduced salt tolerance. These data further suggest that
Zhonghua 11 to 38.10 in oshox22-1. Although the absolute Oshox22 functions as a negative regulator in drought and
ABA level was lower in the mutant, the level of ABA salt tolerance in rice.
accumulated 38.31 fold whereas this was 21.39 fold in wild
type Dongjin. In the two Oshox22 overexpression lines the
induction level was 15.04 whereas in control Zhonghua 11
plants this was only 9.34, thus not only the absolute ABA Discussion
levels were higher but also the induced levels were higher.
In rice and Arabidopsis, about 50 % of all homeobox genes
Drought and salt responses in oshox22-1 and Oshox22- (Chan et al. 1998; Jain et al. 2008; Mukherjee et al. 2009)
OX plants belong to the HD-Zip family. Although some members
have been implicated in the regulation of development and
To address the function of Oshox22 in drought and salt stress responses, the functions of most HD-Zip genes are
responses, we analysed a homozygous oshox22-1 line and still unknown. Based on mutant and over-expression
two Oshox22-OX lines for drought and salt tolerances. analyses, we here propose a function for the rice HD-Zip

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580 Plant Mol Biol (2012) 80:571–585

Fig. 6 Drought tolerance of

oshox22-1 and Oshox22-OX
plants. a Seedlings of oshox22-1
and Oshox22-OX before
applying the drought-stress.
b After drought stress treatment.
c, d, Survival rates of oshox22-1
and Oshox22-OX plants after
application of drought stresses.
e Water-loss rates in leaves
from oshox22-1 and
corresponding wild type plants.
f Water-loss rate in leaves from
Oshox22-OX and corresponding
wild type plants

family I gene Oshox22 in regulating ABA biosynthesis and gene expression without the requirement of exogenous
ABA-mediated drought and salt tolerances in rice. activation domains. Further functional analysis in yeast
In this study, we used a GFP-tagged fusion construct to showed that both HD and Zip domains are required for the
demonstrate that Oshox22 is a nuclear-localized protein, trans-activation. The function of Oshox22 as a transcrip-
which is consistent with a function as transcription factor. tional activator was further confirmed with transient assays
The DNA-binding and activation properties were analysed in in rice protoplasts using a GUS reporter gene. Sessa et al.
yeast one-hybrid experiments, showing specific binding to a (1993, 1997) propose that AH1 (CAAT(A/T)ATTG) and
known HD-Zip target sequence and activation of reporter AH2 (CAAT(C/G)ATTG) act as consensus binding sites for

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Plant Mol Biol (2012) 80:571–585 581

et al. 2002). ABI1 is involved in various responses towards

ABA including stomata closure, seed dormancy and veg-
etative growth and thus represents a key component in
ABA signaling (Leung et al. 1997; Leung and Giraudat
1998). For Athb-7 and Athb-12, it has been found that their
expression is down-regulated in abi1 mutants (Olsson et al.
2004), which further supports the interaction between ABA
signaling and these HD-Zip genes. To perform functional
analyses, we generated Oshox22 over-expression lines and
obtained an oshox22 mutant that contains a T-DNA
insertion in the 50 upstream sequence of Oshox22 and
displays strongly down-regulated expression of this gene.
Under normal greenhouse conditions, oshox22-1 plants did
not show any visible difference from wild type. However,
the endogenous ABA levels in oshox22-1 seedlings were
60 % lower than those of the wild type Dongjin plants.
When grown on medium with ABA, oshox22-1 seeds
showed a higher germination rate than the wild type. On
the other hand, rice plants over-expressing Oshox22
showed a decreased germination rate on medium supplied
with ABA suggesting Oshox22 is involved in an ABA-
regulated seed germination process. It should be noted that
the over-expression and mutation phenotypes were ana-
lysed in different genetic backgrounds. Zhonghua 11 is an
important lowland rice cultivar in Chinese agriculture
which is easy to transform using A. tumefaciens and
Fig. 7 Stomata counting in leaves from oshox22-1 and Oshox22-OX therefore this is the preferred model for transgenesis in our
plants. a The number of stomata on oshox22-1 leaves compared to laboratory. As a mutant allele of Oshox22 was not present
wild type (P \ 0.01, n = 10). b The number of stomata on two in the Zhonghua 11 RMD T-DNA collection (Zhang et al.
independent Oshox22-OX lines compared to wild type (P [ 0.05, 2006), we analysed a mutant allele of Oshox22 in Dongjin,
n = 10). Dongjin in this context is the wild type segregated from
heterozygous oshox22-1/Oshox22 line; ZH11 (Zhonghua 11), wild which is also a lowland Japonica rice cultivar. Since ABA
type segregated from an Oshox22-OX T1 line sensitivity was decreased upon Oshox22 mutation in
Dongjin background and increased upon over-expression in
Zhonghua 11 background, our data suggest that the func-
HD-Zip I and HD-Zip II proteins, respectively. Apparently, tion of Oshox22 between these cultivars is conserved.
Oshox1 to Oshox7 follow these rules (Meijer et al. 1997, These results of Oshox22 effects on ABA sensitivity are
1998, 2000b). Athb-7 and Athb-12, however, do not seem to only partly consistent with those obtained in Arabidopsis
bind to either of these consensus sequences and may have with Athb-7 and Athb-12, in spite of the fact that the latter
totally different binding preferences (Himmelbach et al. are in the same HD-Zip I subgroup (c-clade) as Oshox22
2002; Deng et al. 2006). Our data show that Oshox22 is able (Olsson et al. 2004; Henriksson et al. 2005; Agalou et al.
to activate gene expression via both AH1 and AH2, but more 2008). Furthermore, over-expression of CpHB-7 isolated
effectively via AH2 than AH1. Taken together, our data from from C. plantagineum in Arabidopsis resulted in increased
the yeast and protoplast experiments support the function of germination on ABA and thus reduced sensitivity towards
Oshox22 as a transcriptional activator, which is typical of ABA (Deng et al. 2006). Plants over-expressing Athb-6 are
HD-Zip I family transcription factors (Ohgishi et al. 2001; also less sensitive to ABA on germination (Himmelbach
Meijer et al. 2000b). et al. 2002). Thus, different HD-Zip proteins may be
Similar to closely related HD-Zip factors such as Athb- involved in different ABA-mediated signaling pathways
6, Athb-7 and Athb-12, our previous (Agalou et al. 2008) and differences of closely related genes in the different
and current work shows that expression of Oshox22 is species suggest that the signaling pathway may have
responsive to drought, salinity and ABA treatments, sug- evolved rapidly.
gesting its role in regulating stress tolerance. In Arabid- Although Oshox22 is induced by ABA, constitutive
opsis, the HD-Zip I protein Athb-6 has been shown to over-expression of Oshox22 in Zhonghua 11, led to an
interact with ABI1, a protein phosphatase 2C (Himmelbach increased ABA level but compromised drought tolerance.

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582 Plant Mol Biol (2012) 80:571–585

Fig. 8 Examinations of salt

tolerance of oshox22-1 and
Oshox22-OX plants.
a Seedlings of oshox22-1 and
Oshox22-OX before salt
treatments. b Plants treated with
150 mM NaCl for 9 days. c,
d Green leaf rates for oshox22-
1, Oshox22-OX and their wild
type plants after salt treatments

In addition to decreasing ABA sensitivity, however, the catabolic genes (e.g. ABA 80 -hydroxylase). Furthermore,
Oshox22 mutation in Dongjin resulted in an increased wheat TaABA80 OH1 deletion lines that accumulate higher
tolerance towards drought, which correlated with reduced levels of ABA in spikes are drought sensitive (Ji et al.
water-loss efficiencies of mutant seedlings. As numbers of 2011). We analysed ABA levels in oshox22-1 mutants and
stomata were decreased in oshox22-1 mutants, regulation the two over-expression lines as well as their respective
of stomata density by Oshox22 is a possible mechanism wild type backgrounds. We found that mis-expression of
underlying enhanced drought tolerance in these mutants. Oshox22 affected the absolute levels of ABA. However,
However, a change in stomata density was not found in the despite difference in induction levels, in all plants ABA
Oshox22-OX lines although these lines were less drought levels increased strongly in response to drought, which is in
tolerant. Therefore, most likely stomata density is not the line with the well-known function of this hormone in
only factor determining drought tolerance under our responses to stress signals including drought and salinity.
experimental conditions. We conclude that Oshox22 neg- Higher levels of ABA are due to induction of ABA bio-
atively regulates drought tolerance in rice. In wheat a synthesis genes and in turn ABA reprograms plant cells to
similar situation is described where the level of ABA is withstand and survive adverse environmental conditions
inversely correlated to the level of drought tolerance. In (reviewed by Leung and Giraudat 1998; Umezawa et al.
drought-sensitive cultivars, drought treatment leads to 2009; Melcher et al. 2010; Huang et al. 2012). We
enhanced expression of ABA biosynthesis genes in anthers hypothesize that Oshox22 may plays a role in ABA bio-
and ABA accumulation in spikes, while in drought-tolerant synthesis or degradation and that a lower endogenous ABA
wheat the treatment leads to accumulation of lower levels level in oshox22-1 plants and higher level of ABA,
of ABA, which correlates with lower expression of ABA respectively correlate inversely with their drought toler-
biosynthesis genes and higher level of expression of ABA ance. Being a transcription factor, Oshox22 may affect

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Plant Mol Biol (2012) 80:571–585 583

ABA level by directly regulating genes involved in bio- Acknowledgments We acknowledge the support from projects
synthesis or degradation of ABA. CEDROME (INCO-CT-2005-015468) for PBFO and CML, the
National Natural Science Foundation of China (30821007) and the
We propose that Oshox22 functions as a negative reg- CAS/SAFEA International Partnership Program for Creative
ulator in drought and salt tolerance similar to OsABI5, Research Teams (20090491019) for SZ and CML, TF-STRESS
which is a bZIP transcription factor, and is inducible by (QLK3-CT-2000-00328) for AHM, RNA Seed from the Royal
ABA and high salinity (Zou et al. 2008). Transgenic rice Netherlands Academy of Arts and Sciences (KNAW-CEP
08CDP036) for SZ, PBFO and HS, from the Higher Education
plants over-expressing OsABI5 were sensitive to ABA and Commission (HEC) Pakistan to IH and the Netherlands Organization
to high-salinity stress as well as to PEG treatment. Similar for Scientific Research (NWO; VICI-grant to HB). We thank Francel
as with the oshox22-1 mutant, down-regulation of OsABI5 Verstappen for his technical support at WUR in The Netherlands.
in plants using an RNAi approach exhibited increased
tolerance to salt as well as to PEG treatment which is a
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