research paper seven zinc-ﬁnger transcription factors are

Journal of Experimental Botany, of 14doi:10.1093/jxb/ers035

RESEARCH PAPER

Seven zinc-finger transcription factors are novel regulatorsof the stress responsive gene OsDREB1B

Duarte D. Figueiredo, Pedro M. Barros, Andre M. Cordeiro, Tania S. Serra, Tiago Lourenco, Subhash Chander,

M. Margarida Oliveira and Nelson J. M. Saibo*

1 Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnologica, Av. daRepublica, 2780–157 Oeiras, Portugal

* To whom correspondence should be addressed. E-mail: [email protected]

Received 14 July 2011; Revised 20 January 2012; Accepted 25 January 2012

Abstract

Plants have evolved several mechanisms in order to cope with adverse environmental conditions. The transcription

factors (TFs) belonging to the DREB1/CBF subfamily have been described as major regulators of the plantresponses to different abiotic stresses. This study focused on the rice gene OsDREB1B, initially described as highly

and specifically induced by cold. However, here it is shown that OsDREB1B is not only induced by low temperatures,

but also by drought and mechanical stress. In order to identify novel TFs that bind to its promoter, a yeast one-

hybrid system was used to screen a cold-induced cDNA expression library. Thereby seven novel Zn-finger TFs were

identified that bind to the promoter of OsDREB1B. Among them, there were four Zn-finger homeodomain (ZF-HD)

and three C2H2-type Zn-finger TFs. Gene expression studies showed that these TFs are differentially regulated at

transcriptional level by different abiotic stress conditions, which is illustrative of the crosstalk between stress

signalling pathways. Protein–protein interaction studies revealed the formation of homo- and heterodimers amongthe ZF-HD TFs identified, but not for the C2H2-type. Using a transactivation assay in Arabidopsis protoplasts, all the

TFs identified repressed the expression of the reporter gene, driven by the promoter of OsDREB1B. This assay also

showed that the dimerization observed between the ZF-HD TFs may play a role on their transactivation activity. The

results here presented suggest a prominent role of Zn-finger TFs in the regulation of OsDREB1B.

Key words: Abiotic stress, C2H2, cold, DREB1/CBF, drought, mechanical stress, salt, ZF-HD, Zn finger.

Introduction

Given their sessile nature, plants have adapted to cope with

environmental stresses, such as extreme temperatures,

drought, and salinity. Many of these adaptations take place

at the molecular level and are modulated by transcription

factors (TFs), mediating the stress responses. The APE-TALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF)

family of TFs plays a prominent role in the response to

abiotic stress conditions, particularly through the action of

the DROUGHT RESPONSE ELEMENT BINDING 1/C-

REPEAT BINDING FACTOR (DREB1/CBF) subfamily

(Gilmour et al., 1998; Liu et al., 1998). Elements of this

subfamily were first identified in Arabidopsis, but are now

known to be present in many other plant species, such asrice (Dubouzet et al., 2003), maize (Qin et al., 2004), grape

(Xiao et al., 2006), and cotton (Shan et al., 2007), which

illustrates their relevance in plant stress signalling. Even

though several genes have been identified as targets of

DREB1/CBFs (Seki et al., 2001; Fowler and Thomashow,

2002; Maruyama et al., 2004), there is still much to learnabout their upstream regulators. ICE1 from Arabidopsis

was the first protein identified as a regulator of DREB1/CBF

gene expression, through the binding to a specific cis-motif

present in the promoter region of DREB1A (Chinnusamy

et al., 2003). Afterwards, MYB15 was also described as

interacting with ICE1 and binding to the promoters of

Arabidopsis DREB1/CBFs, working to repress their tran-

scription (Agarwal et al., 2006). More recently, CAMTA3was shown to be a positive regulator of Arabidopsis

ª The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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DREB1C/CBF2, through its binding to the CM2 cis-motif,

present in the promoter of that gene (Doherty et al., 2009),

while PIF7 was established as a possible link between the

light and cold signalling pathways, since it binds to the

promoter of DREB1B and DREB1C (Kidokoro et al., 2009)

and is a negative regulator of phytochrome signalling (Leivar

et al., 2008). Nevertheless, other than in Arabidopsis, very

little is known about the regulation of DREB1/CBFs. Thecurrent work focuses on the rice gene encoding OsDREB1B,

which was initially described as highly and specifically

induced by low temperatures (Dubouzet et al., 2003), but

later, it was found to also respond to high salinity, osmotic

stress, and salicylic acid (Gutha and Reddy, 2008).

There is much interplay in the stress signalling pathways,

namely between different families of TFs. One group of TFs

that has been described as interplaying with DREB1/CBFs,and having a major role in abiotic stress signalling, is the

Zn-finger TFs. Among the different families of Zn-finger

TFs, the C2H2-type were described as involved in several

stress signalling pathways (Sakamoto et al., 2004; Mittler

et al., 2006; Huang et al., 2007; Xu et al., 2008). These TFs,

also referred to as the TFIIIA-type finger, are characterized

by two Cys and two His residues that bind to a zinc ion

(Pabo et al., 2001). Among the members of this class, ZAT12was described as a negative regulator of the DREB1/CBF

regulon (Vogel et al., 2005), while the ZAT10/STZ gene

expression was shown to be dependent on DREB1A/CBF3

(Maruyama et al., 2004). C2H2-type TFs are therefore

signalling components that can be located either up- or

downstream of the DREB1/CBF genes. Another class of Zn-

finger TFs that has been implicated in abiotic stress signalling

is the Zn-finger homeodomain proteins (ZF-HD), whichare characterized by the presence of Zn-finger-like motifs

upstream of a homeodomain (Windhovel et al., 2001).

Interestingly, coexpression of the stress-inducible ZFHD1

and NAC transcription factors enhances the EARLY

RESPONSIVE TO DEHYDRATION1 (ERD1) gene expres-

sion in Arabidopsis (Tran et al., 2006). Still, there are no

reports on the role of the ZF-HD in the responses to abiotic

stress in rice.In this work, using a yeast one-hybrid (Y1H) system,

seven Zn-finger TFs, three C2H2-type, and four ZF-HD,

were identified as binding to the promoter of the rice gene

OsDREB1B. The genes coding for these TFs are differen-

tially regulated by several abiotic stress conditions. More-

over, all these TFs function as transcription repressors and

only the ZF-HD can form homo- and/or heterodimers. This

study reports for the first time the direct regulation ofa DREB1/CBF gene by Zn-finger TFs.

Materials and methods

Plant materials and construction of the cold-induced cDNA

expression library

Eight-day-old rice seedlings (Oryza sativa L. cv. Nipponbare)grown at 28 �C and 12/12 light/dark photoperiod, were subjectedto an 8 �C treatment. Whole seedlings were sampled after 2, 5, and

24 h of cold treatment. RNA was extracted using the RNeasyPlant Mini Kit (Qiagen). Total RNA samples from the three timepoints were pooled in equal amounts for mRNA purification usingthe PolyATtract mRNA Isolation System IV (Promega). Anunidirectional cDNA expression library was prepared in kACTIIusing the HybriZAP-2.1 XR cDNA Synthesis Kit and theHybriZAP-2.1 XR Library Construction Kit (Stratagene), accord-ing to the manufacturer’s instructions. Three lg mRNA were usedto perform the synthesis of first strand cDNA. In vivo massexcision of pACTII’ phagemid from kACTII was performed asdescribed (Ouwerkerk and Meijer, 2001).

Yeast one-hybrid screening

The promoter of OsDREB1B was divided into four overlappingfragments to be used as baits for the Y1H screening. Fragmentlengths and primer pairs used can be found in SupplementaryTable S1 (available at JXB online). The fragments were cloned inthe pHIS3/pINT1 vector system (Ouwerkerk and Meijer, 2001)and integrated into the Y187 yeast strain (Clontech). The baitstrains were transformed with the cDNA expression library. Foreach promoter fragment, over one million yeast colonies werescreened in CM-His medium supplemented with 5 mM 3-amino-1,2,4-triazole, as described (Ouwerkerk and Meijer, 2001). Theplasmids from the yeast clones that actively grew on selectivemedium were extracted and the cDNA insert sequenced. Thesesequences were used to search for homology in the rice genome,using the BLAST algorithm. Plasmids containing genes encodingtranscription factors were re-transformed into the respective baitstrain, to confirm activation of the reporter HIS gene.

Abiotic stress treatments

Rice seedlings were grown hydroponically in rice growth medium(Yoshida et al., 1976) at 28 �C, 700 lmol photons m�2 s�1, 70%humidity, and 12/12 light/dark photoperiod for 14 days. Theseedlings were then transferred to stress conditions 4 h after dawn.At the same time, control seedlings were transferred to freshgrowth medium (mock control). Cold treatments were performedby transferring the seedlings to growth chambers at either 5 �C or10 �C in pre-cooled medium. For salt and abscisic acid (ABA)treatments, seedlings were transferred to growth media supple-mented with 200 mM NaCl or 100 lM ABA, respectively.Drought treatment was performed by maintaining the seedlingsover dry absorbent paper. The mechanical stress assay was carriedout by damaging the seedlings before they were transferred to newgrowth medium. All other conditions were maintained throughoutthe assays. Ten plants were sampled for each time point, and rootsand shoots were harvested separately. All assays were repeated atleast twice.

Semi-quantitative reverse-transcription PCR

Total RNA was extracted using the RNeasy Plant Mini Kit(Qiagen). First strand cDNA was synthesized from 1 lg totalRNA, using an oligo-dT primer (Invitrogen) and the SuperscriptIIreverse transcriptase (Invitrogen), following the manufacturer’sinstructions. The gene-specific primers, as well as the number ofPCR cycles and annealing temperature used for each pair, canbe found in Supplementary Table S2. The amplification wasperformed using GoTaq polymerase (Promega) with 2.5 mMMgCl2, 12.5 lM of each primer, and 0.2 mM dNTPs. In the casesof OsZHD4 and of OsZHD8, 5% DMSO was added to the finalmix. ACTIN1 (Os03g50885) was used as an internal control for allexperiments, except for shoots in the drought assay and roots at10 �C, where EUKARYOTIC ELONGATION FACTOR 1-a(Os03g08060) and UBIQUITIN-CONJUGATING ENZYME E2(Os02g42314) were used, respectively. The results shown arerepresentative of at least two biological replicates.

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Transactivation assay

The reporter plasmids were built using the pCAMBIA1391zpromoter-cloning vector backbone, where the kanamycin plantresistance gene, downstream of the full CaMV35S promoter, wasremoved using XhoI and replaced by the Luciferase gene, excisedfrom plasmid pGL3-basic (Promega) with XhoI and SalI. Theminimal CaMV35S promoter (–90 to +8 bp) was cloned upstreamof the GUS gene as a SmaI–EcoRI fragment. This plasmid wasconfirmed by restriction analysis and sequencing, and namedpLUCm35GUS. The OsDREB1B promoter fragments used in theY1H screening were cloned in the pLUCm35GUS reporter vector,using the restriction sites SalI and PstI. Effector plasmids wereconstructed by cloning the coding region of the TFs in pDONR221(Invitrogen), following the manufacturer’s instructions, to obtainthe pENTR-TF. The sequences were then recombined into plasmidpH7WG2 (VIB, Gent), to be under the control of the full CaMV35Spromoter.Arabidopsis protoplasts were prepared as previously described

(Anthony et al., 2004). For each independent transformation, 5 lg ofreporter plasmid and 10 lg of effector plasmid were used. Eachtransformation was performed in triplicate. Cells were incubated for24 h at 22 �C in the dark and then collected at 450 g for 1 min ina swing-out rotor. Cell lysis was performed by resuspension in 150 ll100 mM K2PO4 (from a 1 M pH 7.8 stock solution), 1 mM EDTA(from a 0.5 M pH 8 stock solution), 7 mM 2-mercaptoethanol, 1%Triton X-100, and 10% glycerol, followed by two freeze–thaw cycles.The lysate was then cleared by centrifugation for 2 min at 17,000 g.For the GUS quantification assay, 0.5 ll of 50 mM 4-methylumbel-liferyl-b-D-glucuronide were added to 20 ll of the lysate (intriplicate). Reactions were carried at 37 �C in the dark for 1 h andstopped with 180 ll 200 mM Na2CO3. Fluorescence was detectedusing a spectrofluorimeter (Fluoromax-4 with Micromax platereader, Horiba) with excitation at 365 nm, emission at 455 nm, anda slit of 1.5 nm. Readings were performed in triplicate. Luciferaselevels were determined by adding 150 ll LUC reagent (20 mMTricine pH 7.8, 5 mM MgCl2, 0.1 mM EDTA, 3.3 mM DTT, and2 mM ATP) to 20 ll of the cell lysate. An aliquot (75 ll) of 1.5 mMluciferin was added to each sample and light intensity was readfor 10 seconds in a luminometer (Modulus Microplate, TurnerBiosystems). Readings were performed in triplicate. Activation ofgene expression was calculated as a GUS/LUC ratio.

Yeast two-hybrid

The full coding sequences of the TFs under study were cloned intovector pAD-WT (Stratagene), by replacement of the coding regionof the wild-type lambda cI (fragment C) downstream of the GAL4activation domain, using the enzymes EcoRI and PstI. The TFcoding sequences were also recombined from pENTR-TF (seeabove) into vector pBD-GW. This vector was obtained by cloninga Gateway (GW) cassette into plasmid pBD-GAL4 Cam (Strata-gene). Sense orientation and translational fusion between GAL4-BD- and TF-encoding genes were confirmed by restriction digestionand sequencing, respectively.The pAD and pBD plasmids were transformed into yeast strain

YRG2 (Stratagene), as described (Ouwerkerk and Meijer, 2001).Yeast colonies were plated on CM-Leu-Trp-His media andgrowing colonies were confirmed by PCR. The vectors pAD-WTand pBD-WT (Stratagene) were used as positive control.

Bimolecular fluorescence complementation and cellular localization

For bimolecular fluorescence complementation (BiFC), the codingregions of the TFs under analysis were recombined from plasmidpENTR-TF (see above) into vectors YFPN43 and YFPC43, to bein fusion with the N- and C-terminal portions of yellow fluorescentprotein (YFP), respectively. Cloning was done according toGateway technology (Invitrogen). To use as negative control inthe interaction assays with the TFs, the Arabidopsis SNF1

KINASE HOMOLOG 10 (Akin10) was tested in fusion with theN-terminal portion of the YFP.For cellular localization assays, the coding region of the TFs

were recombined from pENTR-TF into vector pH7WGF2 (VIB,Gent) to be in a translational fusion with GFP and under thecontrol of the full CaMV35S promoter. Cloning was doneaccording to the Gateway technology (Invitrogen). The emptypH7WGF2 vector was used as a negative control for the cellularlocalization.Transformation of Arabidopsis protoplasts was performed as

described for the transactivation assay. For each transformation,3 lg of each plasmid were used. The protoplasts were incubatedovernight in the dark at 22 �C and observed with a fluorescencemicroscope (Leica DMRA2). Nuclear staining was performed byincubating the protoplasts in a 600 ng ll�1 DAPI solution for 2 h.

Results

OsDREB1B transcript level is regulated by severalabiotic stress conditions

The gene expression of OsDREB1B (Os09g35010) was

analysed in plants subjected to several abiotic stress con-ditions, using semi-quantitative reverse-transcription (RT)

PCR. For this, 2-week-old rice seedlings were subjected to

cold (5 and 10 �C), salt (200 mM NaCl), drought, and

ABA (100 lM) treatments for up to 24 h (Fig. 1A and

Supplementary Fig. S1). The results confirmed that

OsDREB1B is highly regulated by cold, as previously

described (Dubouzet et al., 2003). In addition, it was

observed that this regulation is temperature dependent andshows a similar pattern in both shoots and roots (Fig. 1A).

When rice seedlings were subjected to 10 �C, the

OsDREB1B transcript level was rapidly induced (10 min),

reached a peak at 1–2 h, and then started to decrease,

returning to basal levels afterwards. At 5 �C, however, theinduction of OsDREB1B only started after 40 min of cold

and remained high until the end of the assay. Rice seedlings

treated with ABA or subjected to high salinity showeda similar gene expression pattern for OsDREB1B in both

shoots and roots. The transcript level of OsDREB1B was

rapidly (10 min) upregulated after the onset of stress and

followed by a downregulation after 20–40 min. This pattern

was also observed in shoots under drought stress, whereas

in roots the transcript level of OsDREB1B was kept high at

least during 24 h after drought treatment. This suggests that

OsDREB1B may play an important role in the plantresponse to drought, particularly at root level. In the case

of NaCl and ABA treatments, there was also a transient

upregulation of OsDREB1B after 5–10 h of NaCl treatment

in shoots and 1–2 h of ABA treatment in roots. However,

given that these changes also appear in the mock control,

they are likely to be not specific to NaCl and ABA

treatments.

Under mock treatment, a circadian regulation ofOsDREB1B could be observed, with the transcript level

reaching a peak at 2–5 h after the start of the assay (6–9 h

after dawn), decreasing afterwards. In addition, under the

same treatment, OsDREB1B showed a transient increase of

gene expression at 10 and 20 min. Since, in this case, the

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only change in conditions was the transfer of the plants to

new growth medium, it was hypothesized if this upregula-

tion could be due to a response to mechanical stress.Therefore, another assay was performed, in the same

conditions as above, but where the plants were damaged

and transferred to new medium. In this case, the

OsDREB1B transcript level was highly induced in response

to mechanical damage for at least 20 minutes, returning to

basal levels afterwards (Fig. 1B). This may also explain the

transient upregulation that was observed in the salt,

drought, and ABA treatments around 10 and 20 min afterthe start of stress (Fig. 1A and Supplementary Fig. S1).

Nevertheless, the transient induction observed in the salt

and drought treatments seems to be more significant than

the one seen in the mock treatment.

Seven novel Zn-finger TFs identified as binding to thepromoter of OsDREB1B

In order to identify TFs that bind to the promoter of

OsDREB1B, a Y1H screening was performed, using that

promoter as bait. The 2000 bp upstream of the OsDREB1B

ATG start codon were considered to be the promoter

region. A bioinformatics prediction of TF binding sites and

other cis-regulatory elements in this sequence can be found

in Supplementary Table S3. For the Y1H assay, this 2000

bp sequence was divided into four overlapping fragments of

around 500 bp, numbered from 1 to 4 (Supplementary

Table S1), 1 being the fragment further away from the start

codon and 4 the one closest to it (Fig. 2A). The four yeastbait strains (each fragment corresponds to one bait strain)

were used to screen a rice cold-induced cDNA expression

library, and at least one million yeast colonies were screened

for each fragment. This allowed the identification of seven

Zn-finger TFs and of a bHLH TF (Figueiredo et al.,

unpublished results) as binding to the OsDREB1B pro-

moter. Fig. 2A shows the relative position of the Zn-finger

TFs identified along the promoter of OsDREB1B. Four ZF-HD TFs were found as interacting with the DNA sequence

between –1527 to –961 bp upstream of the OsDREB1B start

codon, one C2H2-type Zn-finger TF was found between

–1028 to –388 bp, and two other C2H2-type Zn-finger TFs

were found between –488 bp and –3 bp. No TFs were found

interacting with the promoter fragment ranging from –1945

to –1447 bp upstream of the start codon. In order to

confirm the protein–DNA interactions, the yeast bait strainswere then re-transformed with the plasmids containing the

TF coding sequences. The identified TFs were named

according to previous studies (Agarwal et al., 2007; Hu

et al., 2008). Table 1 shows the name, gene locus, and

number of times each TF was identified in the screening.

Fig. 2B shows the protein domains identified in each of the

TFs under study. The ZF-HD TFs had a protein sequence

Fig. 1. Transcriptional profile of OsDREB1B in rice seedlings subjected to different stress treatments. (A) OsDREB1B gene expression

analysed by semi-quantitative RT-PCR in plants subjected to cold (5 and 10 �C), ABA (100 lM), NaCl (200 mM), drought, and mock

control. All treatments started (time 0) at 4 h after dawn. Roots and shoots were analysed separately. The internal control for each assay

is described in the Material and methods section. (B) OsDREB1B gene expression analysed by semi-quantitative RT-PCR in plants

subjected to mechanical stress. Whole plants were assayed.



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with the ZF-HD upstream of the C-terminal DNA-binding

homeodomain, as previously described (Windhovel et al.,2001). OsZHD1 and 2 had very similar domain structures

with the homeodomain close to the C-terminus, whereas

in OsZHD4 both domains were more central in the

protein. In the case of ZHD8, the homeodomain was

localized similarly to the one on OsZHD4, but the ZF-

HD domain was closer to the N-terminal of the protein,

when compared to the other ZF-HD TFs. As for the

C2H2-type TFs, ZOS12-7 was found to have three Zn-fingers, while the other two, ZOS3-12 and ZOS11-10,

only had two. ZOS3-12 showed an additional feature at

its C-terminus: a DLN-box/EAR-motif, which was de-

scribed as a transcriptional repressor domain (Ohta et al.,2001). No such motifs were identified in the other TFs

under study. Phylogenetic analysis comparing the sequen-

ces of rice and Arabidopsis C2H2 and ZF-HD TFs can be

found in previous studies (Agarwal et al., 2007; Hu et al.,

2008). Supplementary Fig. S2 shows an alignment of the

DNA-binding domains of the Zn-finger TFs identified in

this study with the homologous proteins previously

described in Arabidopsis.In order to test whether the TFs identified bind specifi-

cally to the promoter fragment used as bait in the Y1H

Table 1. Zn-finger transcription factors identified as binding to the promoter of OsDREB1B

RGAP, Rice Genome Annotation Project; ZF-HD, Zn-finger homeodomain.

Promoter fragment (bp) RGAP gene locus Conserved domains Name No. of times identified Reference

From –1527 to –961 Os09g29130 ZF-HD OsZHD1 1 Hu et al. (2008)

Os08g37400 ZF-HD OsZHD2 2



From –1028 to –388 Os12g38960 C2H2 ZOS12-7 2 Agarwal et al.(2007)

From –488 to –3 Os03g32230 C2H2 ZOS3-12 1

Os11g47630 C2H2 ZOS11-10 1

Fig. 2. Transcription factors binding to the OsDREB1B promoter. (A) Schematic representation of the OsDREB1B promoter, divided into

the four fragments used in the yeast one-hybrid screening, and the transcription factors (TFs) identified. The oval shapes represent the

TFs identified as binding to each of the promoter fragments. White, Zn-finger homeodomain TFs; grey, C2H2-type Zn-finger TFs.

Positioning of the TFs along each promoter fragment is not indicative of the position of their binding sites and is only based on yeast one-

hybrid screening. (B) Schematic representation of the protein domains of each TF. Dark grey, Zn-finger homedomain; light grey,

homeodomain, striped, zinc-fingers; black, DLN-box/EAR-motif.



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screening, each yeast bait strain was transformed with the

seven Zn-finger TFs, independently. Fig. 3 shows that none

of the TFs under study activated the expression of the HIS

reporter gene when transformed into the bait strain carrying

the OsDREB1B promoter fragment ranging from –1975 to

–1447 bp. In addition, the ZF-HD TFs bound only to the

promoter fragment ranging from –1527 to –961 bp, which

correlates to the fact that this promoter region is the onemost enriched in ZF-HD binding motifs (Supplementary

Fig. S3; Tan and Irish, 2006). Regarding the C2H2-type

TFs, ZOS12-7 bound only to the –1028 to –388 bp

promoter region, while ZOS3-12 and ZOS11-10 bound to

the one ranging from –488 to –3 bp, as predicted by the

Y1H screening results, but also to the fragment –1028 to

–388 bp. Altogether, these results showed that the binding

of these TFs is specific to the promoter fragments used andnot to the backbone vector. The fact that ZOS3-12 and

ZOS11-10 bound to both promoter fragments (–1028 to

–388 bp and –488 to –3 bp), correlates with the fact that

both fragments have binding sites for these two TFs. There

are several reasons why these TFs were initially not

identified through the Y1H screening as binding to –1028

to –388 bp (Fig. 2). Among others, they may have higher

affinity to fragment –488 to –3 bp or they may have highertranscriptional activity when bound to this fragment as

compared to fragment –1028 to –388 bp. Interestingly, the

promoter fragment ranging from –1028 to –388 bp shows

an enrichment in the A(G/C)T-X3-4-A(G/C)T cis-regulatory

element (Supplementary Fig. S3), which had previously

been described as a binding site for C2H2-type TFs in

Arabidopsis (Sakamoto et al., 2004). The observation that

ZOS12-7 only interacts with the promoter fragment rangingfrom –1028 to –388 bp, suggests that it has a different

DNA-binding specificity when compared to ZOS3-12 and

ZOS11-10. This is supported by the fact that the DNA-

binding domain of ZOS12-7 shares the least similarity when

compared to ZOS3-12, ZOS11-10 and to the Arabidopsis

TFs described by Sakamoto et al. (2004) (Supplementary

Fig. S2).

The genes encoding the Zn-finger TFs identified aredifferentially regulated under abiotic stress

To understand whether the seven Zn-fingers identified havea role in the plant response to abiotic stress conditions, their

gene expression was analysed by semi-quantitative RT-PCR

in rice seedlings subjected to the conditions previously

tested for OsDREB1B (Fig. 4). The most relevant gene

expression profiles were quantified and normalized to the

internal controls (Supplementary Fig. S4). All genes under

analysis showed a constitutive basal expression in both

roots and shoots, and the mock control revealed that somegenes undergo a circadian rhythm regulation. This is most

striking for genes OsZHD2 and ZOS3-12 in roots and

ZOS11-10 in roots and shoots.

Regarding the four ZF-HD genes, their expression in

shoots was not significantly or consistently altered

Fig. 3. Analysis of the DNA-binding specificity of the Zn-finger

transcription factors (TFs) identified. All the four yeast bait strains

used in the Y1H screening were transformed with the seven

different TFs fused to the GAL4 activation domain. The left

column (–HIS–LEU) shows the selection plates, indicating

positive interactions between the TFs and the respective

promoter fragments. The right column (+HIS–LEU) shows

the positive controls (yeast bait strains grown on histidine-

supplemented medium). The bottom diagram indicates the

TFs position on each plate.



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Fig. 4. Transcriptional profile of the transcription factors (TFs) identified as binding to the promoter of OsDREB1B. The transcriptional

profile was obtained by semi-quantitative RT-PCR for plants subjected to cold (5 and 10 �C), ABA (100 lM), NaCl (200 mM), drought,

and mock control. All treatments started (time 0) at 4 h after dawn. Genes used as internal controls for each assay are described in the

Materials and methods section.



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throughout most treatments. There were however some

exceptions, like the induction of both OsZHD4 at 5 �C and

of OsZHD8 by high salinity and drought. We also

observed a repression of OsZHD2 and OsZHD4 2 h after

drought and of OsZHD8 10 min after ABA treatment.

Nevertheless, for most stress assays, the changes in ZF-HD

gene expression in shoots were not significant. The same

does not apply however to root transcription profiles. The

transcript level of OsZHD1 in roots seemed to be

transiently induced by cold (only at 5 �C) and by drought.

The expression of gene OsZHD2, on the contrary, was

induced by all stress conditions, even if only for a short

period, followed by a return to basal levels. OsZHD4 was

mainly induced by ABA, NaCl and drought, but not bycold. OsZHD8 showed an increase in expression under

several stress conditions – cold, ABA and drought, with

a return to basal levels at the later stages of the treatments.

Concerning the C2H2-type Zn-finger TFs, the three genes

showed very distinct expression patterns. The ZOS3-12

transcript level was rapidly and highly upregulated in

response to all the stress conditions tested, both in roots and

in shoots (except for ABA in roots). The transient inductionof this transcript after 10 min in the mock control suggests

a response to mechanical stress, as observed for OsDREB1B

(Fig. 1). Therefore, the rapid induction (10 – 20 min) upon

10 �C and ABA might be biased. Interestingly, and similarly

to OsDREB1B, the ZOS3-12 transcript level is more rapidly

induced at 10 �C than at 5 �C. The ZOS11-10 gene

expression seemed to be particularly induced under cold in

both shoots and roots. The induction is later in the roots, but

the transcripts reached higher levels. ZOS11-10 was slightly

induced under salt (roots) and drought (roots and shoots),

but this regulation might be biased by the circadian rhythm

observed in the mock control. In roots, this gene was

downregulated in response to ABA treatment. Regarding

ZOS12-7, it was late (5 – 10 h) and transiently induced inshoots by ABA and in roots it was induced at 10 �C (after

10 h treatment).

The Zn-finger-HD, but not the C2H2-type, TFs formhomo- and heterodimers

To test whether the TFs under study would form homo-

or heterodimers, and also if they would interact with one

another, a direct yeast two-hybrid (Y2H) assay was

performed. Each TF was tested for interactions with itselfand with the other six TFs under study. As shown in Fig.

5, only interactions for the ZF-HD TFs could be

detected, not for any of the C2H2-type TFs under study.

The only TFs that were shown to homodimerize in this

assay were OsZHD1 and OsZHD4. These two proteins

also interacted in yeast with all the other ZF-HD TFs

under study, whereas OsZHD2 and OsZHD8 only inter-

acted with OsZHD1 and with OsZHD4, but not witheach other.

In order to confirm these interactions, a BiFC assay was

performed with the TFs that yielded positive results for the

Y2H. Using this system all the interactions previously

observed could be validated (Fig. 6). In all cases, the

Fig. 5. Direct yeast two-hybrid assay to test interactions between the Zn-finger transcription factors (TFs). TFs were cloned in

fusion with both the GAL4-AD and GAL4-BD and all the plasmid combinations were transformed into the yeast strain YRG2. Left

panel: yeast growth on –His–Leu–Trp selection medium. Right panel: yeast growth on +His–Leu–Trp selection medium. Positive

control used was the interaction of pAD-WT and pBD-WT. The selection medium for the interactions of pAD-ZOS3-12 and pAD-

ZOS11-10 was supplemented with 5 mM 3-amino-1,2,4-triazole, whereas for pAD-OsZHD2, 10 mM 3-amino-1,2,4-triazole was

used.



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fluorescence signal was clearly localized, most likely in the

nucleus of the cell. This is supported by the nuclear

localization of all the individual TFs under study (Supple-

mentary Fig. S5). For some of the interactions several spots

of fluorescence could be seen, in what could be nuclear

bodies.

The Zn-finger TFs binding to the OsDREB1B promoterhave repressor activity

In order to determine whether the TFs identified as binding

to the promoter of OsDREB1B were repressors or activators

of gene expression, transactivation assays were performed in

Arabidopsis protoplasts. Different reporter vectors were used

in which the GUS gene is under the control of the minimal

CaMV35S promoter plus the respective OsDREB1B pro-

moter fragment that was used in the Y1H screening

(Fig. 7A). Using this strategy, this assay also allowed the

validation of the interactions of the TFs with the promoter

fragments of OsDREB1B.

Regarding the C2H2-type TFs, ZOS3-12 had a predicted

transcriptional repressor DLN-box/EAR-motif domain

(Ohta et al., 2001), but for ZOS11-10 or ZOS12-7 no trans-

acting domains could be predicted. Nevertheless, in Arabi-

dopsis, four C2H2-type TFs (STZ, AZF1, AZF2 and AZF3)

were shown to repress transcription through a A(G/C)T-

X3-4-A(G/C)T cis-element (Sakamoto et al., 2004), which is

present in both OsDREB1B promoter fragments used in the

Y1H screening to identify ZOS11-10 or ZOS12-7 (Supple-

mentary Table S3 and Supplementary Fig. S3). In the study

of Agarwal et al. (2007), a phylogenetic tree of C2H2-type

TFs showed that the rice proteins ZOS3-12, ZOS11-10, and

ZOS12-7 are in the same clade as the Arabidopsis proteins

STZ, AZF1, AZF2, and AZF3. These results, together with

the alignment of the conserved DNA-binding domains

(Supplementary Fig. S2), suggest that these TFs have

similar DNA-binding specificities.

As for the ZF-HD TFs, no predicted repressor or

activator domains identified were present in any of these

proteins. Nevertheless, the results indicated that, in the

conditions tested, all seven Zn-finger TFs are repressors

of gene expression (Fig. 7B). The repressor activity,

measured as a GUS/LUC ratio, ranged from 20% forZOS3-12 to almost 80% for ZOS11-10, which was the

strongest repressor. All the other TFs had intermediate

activities, in the range of 40–60% of the initial GUS/LUC

ratio. Curiously, the only Zn finger having a canonical

repressor domain (ZOS3-12) showed the lowest repressor

activity.

In order to test whether the formation of ZF-HD

heterodimers (Figs. 5 and 6) had an effect on their

transactivation activity, Arabidopsis protoplasts were co-

transformed with all possible combinations of the ZF-HD

TFs binding to OsDREB1B promoter. Fig. 7C shows that

the repression activity observed for OsZHD1, when trans-

formed alone, is no longer observed when this TF is co-

transformed with any of the three TFs with which it forms

heterodimers. In contrast, the co-transformation of

OsZHD2 and OsZHD4 had a clear synergistic effect in

repressing transcription. When OsZHD2 and OsZHD8,

which do not dimerize with one another (Figs. 5 and 6),

were co-transformed, the transactivation activity observed

was similar to the ones of the single TFs. The same was

observed for the co-transformation of OsZHD4 and

OsZHD8, even though these two TFs were observed to

Fig. 6. Bimolecular fluorescence complementation assay to con-

firm protein interactions between the Zn-finger-HD transcription

factors (TFs). On the top panel is the interaction of OsZHD1 with

itself and with the other three ZF-HD TFs that bind to the

OsDREB1B promoter. On the center panel is the interaction of

OsZHD4 with itself and other two Zn-finger-HD TFs. Negative

control used was the interaction of the TFs of interest with the

Arabidopsis Akin10, in fusion with N-terminal portion of YFP as

well as the absence of homodimerization between OsZHD2

(bottom panel).



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heterodimerize and OsZHD4 was shown to forms homo-

dimers (Figs. 5 and 6).

Discussion

The rice gene OsDREB1B was initially reported as specifi-

cally and strongly induced by cold (Dubouzet et al., 2003)

and shown to be also regulated, to some extent, by salt,osmotic, and oxidative stresses and salicylic acid (Gutha

and Reddy, 2008). In the work here presented, it was

observed that OsDREB1B gene expression is indeed highly

induced by low temperature and that this induction is

dependent on the intensity of the stress. Interestingly, the

faster induction of OsDREB1 gene expression at 10 �C, ascompared to 5 �C, was also observed for ZOS3-12 and

ZOS11-10 (Figs. 1 and 4 and Supplementary Figs. S1 andS4). This may be due to the fact that 5 �C is a very severe

stress for rice plants. When subjected to such severe

conditions, rice plants are most likely biochemically im-

paired, resulting in a delay in molecular responses. In our

research group, this response has been observed for other

rice genes (Serra et al., unpublished results). Moreover, it

was also observed that OsDREB1B is very responsive to

drought conditions, but only in the roots. This highlightsthe importance of analysing different tissues separately, in

order not to mask differences in expression between them.

Gutha and Reddy (2008) reported an activation of

OsDREB1B under salt stress that was not observed in the

gene expression studies here presented. The fact that these

authors used a different rice variety may be an explanation

for this difference, but also the experimental designs may

account for some of the gene expression differences. In-terestingly, an early induction of OsDREB1B could be

detected in the mock-treated plants, which turned out to be

a response to mechanical stress. The fact that this gene

responded to the moving of the plants from one flask to

another, i.e. responded to touch, indicates that OsDREB1B

is highly responsive to mechanosensing. This is in agree-

ment with the findings by Gutha and Reddy (2008), who

observed that OsDREB1B transcription is induced inresponse to salicylic acid and that tobacco plants over-

expressing this gene have an enhanced resistance to viral

infection. Moreover, Gilmour et al. (1998) had also

reported an activation of the Arabidopsis DREB1B/CBF1 in

response to mechanical agitation. Therefore, the involve-

ment of this gene in mechanosensing and biotic stress

Fig. 7. Transactivation activity of the transcription factors (TFs)

identified as binding to the OsDREB1B promoter. (A) Constructs

used for Arabidopsis protoplast transformation. Effector constructs

used correspond to the TF coding region under the control of the

full CaMV35S promoter. Reporter constructs contain the GUS

gene driven by the minimal CaMV35S promoter (m35S) plus the

fragment of the OsDREB1B promoter (promOsDREB1B) used as

bait in the Y1H screening. The LUC gene under the control of the

full 35S promoter was used to normalize GUS expression levels.

(B) Transactivation or repression analysis as a GUS/LUC activity

ratio. Each plot refers to a specific OsDREB1B promoter fragment,

as used for the Y1H screening, which is highlighted in the

schemes on top right corners. Values shown are multiples of the

GUS/LUC ratio obtained with the reporter vector without effector.

Values are mean 6 SD (n ¼ 3). *Differences are statistically

significant (t-test, P < 0.05). (C) Analysis of the transactivation

activity for co-transformed ZF-HD TFs. The reporter vector used

was the one carrying the OsDREB1B promoter fragment ranging

from –1526 to –961 bp. Values shown are multiples of the GUS/

LUC ratio obtained with the reporter vector without effector. Values

are mean 6 SD (n ¼ 3). * Differences to the GUS/LUC values of

the reporter vector alone are statistically significant (t-test, P < 0.1).



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responses seems to be conserved in different species. This is

further supported by the fact that the promoter of

OsDREB1B has several putative binding sites for WRKY

TFs (Supplementary Table S3), which are normally associ-

ated with biotic stress signalling. The fact that this study did

not identify any WRKY TF binding to the promoter of

OsDREB1B is probably due to the fact that it used a cDNA

expression library prepared from seedlings subjected to 2, 5,and 24 h of cold. The use of a biotic stress-induced cDNA

expression library would certainly result in the identification

of additional TFs binding to the promoter of OsDREB1B.

Even though the DREB1/CBF regulon has been the

subject of many studies, in several plants, not much is still

known about the TFs that control of expression of these

genes. Phytochrome interacting factor 7 (PIF7) has been

shown to be involved in the regulation of DREB1/CBFs inArabidopsis (Kidokoro et al., 2009). Also, in Arabidopsis, at

least three other TFs were reported to bind to the

promoters of DREB1/CBFs and regulating their transcrip-

tion: ICE1, MYB15, and CAMTA3 (Chinnusamy et al.,

2003; Agarwal et al., 2006; Doherty et al., 2009). In rice

however, no TFs had been identified as regulators of

OsDREBs. In this work, seven Zn-finger TFs, both

homeodomain and C2H2-type, were identified as binding tothe promoter of OsDREB1B. Zn-finger TFs had already

been shown to interplay with DREB1/CBFs in the response

to abiotic stresses, both up- and downstream of the DREB1/

CBF regulon (Maruyama et al., 2004; Vogel et al., 2005).

However, there are no reports on the direct regulation of

DREB1/CBFs by Zn-finger TFs. The fact that in Arabidop-

sis no Zn-finger TFs have been found as binding to the

promoters of DREB1/CBFs, may mean that the regulationof these genes is different in rice and Arabidopsis. Only

a better understanding of the TFs that regulate DREB1/

CBF expression in several plant species will unveil whether

these regulatory pathways are species-specific or not.

Nevertheless, it must also be take into account that the TFs

identified so far, as regulating DREB1/CBFs, may only be

a small fraction of the total TFs involved in the regulation

of these genes.The gene expression of ZF-HD and C2H2-type TFs has

been already described as regulated by abiotic stress

conditions in Arabidopsis (Sakamoto et al., 2004; Tan and

Irish, 2006) and in rice (Agarwal et al., 2007; Jain et al.,

2008). In the current study, the gene expression of some TFs

was highly altered by all stresses applied (such as ZOS3-12),

while for others the transcript level did not show significant

variations (like OsZHD1), and others responded specificallyto one particular type of stress (such as ZOS12-7, both in

shoots and roots). Again, and similarly to what happens

with OsDREB1B, for some of the genes, major differences

could be observed between expression patterns in roots and

shoots, indicating that stress responses are tissue-specific. In

Arabidopsis, it has been observed that several genes are

differentially regulated in shoots and roots in response to

several abiotic stress conditions (Kreps et al., 2002; Kilianet al., 2007). The fact that the expression of genes encoding

regulators of OsDREB1B is modulated by these many

environmental conditions is indicative of the crosstalk

between different stress signalling pathways, where

OsDREB1B seems to play an important role.

Microarray data are already available on the expression

profiles of rice ZF-HD TFs under abiotic stress conditions

(Jain et al., 2008). These authors observed an upregulation

of OsZHD1 in drought and of OsZHD4 in cold, which

correlates with the data here presented. Regarding theC2H2-type TFs, the expression data in this study for

ZOS3-12, indicating an induction by several types of

stress, also correlate with previous microarray data that

reported its upregulation by cold, salt, and drought

(Agarwal et al., 2007). In that study, the authors had also

observed an induction of ZOS11-10 by cold, similarly to

what is observed here, both in roots and shoots. The fact

that a time course up to 24 h was analysed here, instead ofsingle time point, as performed by Jain et al. (2008) and

Agarwal et al. (2007), may explain why some gene

expression responses described here were not observed in

those studies.

It was also interesting to note that, even though the

Zn-finger TFs identified were shown to be negative

regulators of OsDREB1B transcription, their gene expres-

sion pattern under cold is, in some cases, very similar tothe one of OsDREB1B. This is particularly striking in

the case of ZOS3-12 (Figs. 1 and 3 and Supplementary

Figs. S1 and S4). Nevertheless, the increased ZOS3-12

transcript level, caused by either 5 or 10 �C treatment and

observed in both roots and shoots, starts always later than

the increase observed for OsDREB1B transcript level. The

fact that the induction of ZOS3-12 upon 5 �C treatment

does not seem to have an immediate negative effect on thetranscript levels of OsDREB1B may be due to competition

with other TFs that activate the expression of OsDREB1B.

In addition, it must be noted that an increase in gene

expression does not always correlate directly with an

increase in protein levels.

This study also tested whether the Zn-finger TFs identi-

fied interact with themselves and one another. Interactions

were only observed between the ZF-HD TFs. The C2H2-type TFs did not interact with one-another, nor with the

ZF-HD TFs, in yeast. Homo- and heterodimers had already

been described for ZF-HD TFs in Arabidopsis (Tan and

Irish, 2006) and in Flaveria (Windhovel et al., 2001),

meaning that this feature is conserved in several species for

this type of TFs. The heterodimerization of two homeodo-

main proteins from the mushroom Coprinus cinereus was

shown to be necessary for their function as transcriptionalregulators, namely in what concerns their targeting to the

cell nucleus (Spit et al., 1998). This is illustrative of the

functional significance of TF heterodimerization.

In this transactivation assay in Arabidopsis protoplasts,

the TFs identified as binding to the promoter of

OsDREB1B all functioned as transcription repressors.

Among previously described ZF-HD TFs, the Arabidopsis

ZHD11/ATHB29/ZFHD1 was described as having tran-scriptional activation activity, while all the other ZF-HD

TFs in this plant did not show such activity (Tan and Irish,



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2006; Tran et al., 2006). It was not determined however if

those TFs would work to repress transcription. Interest-

ingly, the members of this family have been described as

redundant in Arabidopsis (Tan and Irish, 2006), which may

explain the fact that the ZF-HD here identified in rice as

regulators of OsDREB1B all act as in the same way, to

repress its transcription. Regarding the C2H2-type TFs,

different members of this family had already been describedas transcriptional repressors under several abiotic stress

conditions, including cold, in Arabidopsis (Sakamoto et al.,

2004). Moreover, a cis-element present in the OsDREB1B

promoter fragments used in the Y1H screening was de-

scribed as a binding site for Arabidopsis C2H2 TFs (STZ,

AZF1, AZF2, and AZF3) and to act as a negative regulator

of transcription (Sakamoto et al., 2004). The fact that the

C2H2-type TFs binding to the OsDREB1B promoter sharehigh sequence similarity with the Arabidopsis STZ, AZF1,

AZF2, and AZF3 (Agarwal et al., 2007), indicates that their

DNA-binding specificity and transactivation activity must

be similar to the one of the Arabidopsis proteins. Neverthe-

less, other C2H2-type TFs involved in abiotic stress

conditions have also been described as transcriptional

activators (Huang et al., 2009; Sun et al., 2010).

The identification of seven transcriptional repressorsbinding to the promoter of OsDREB1 a gene known to be

highly induced by cold (Fig. 1 and Supplementary Fig. S1),

leads to the question of why so many repressors were

identified as binding OsDREB1B promoter. A possible

hypothesis is suggested: when rice seedlings are subjected to

10 �C, the OsDREB1B gene expression is quickly induced,

reaches a peak at 2 h, and then starts to decrease (Fig. 1). In

addition, the cold-induced cDNA expression library usedfor the Y1H screening was prepared from plants subjected

to 8 �C and collected 2, 5, and 24 h after the start of the

stress (see Materials and methods). Thus, if at 8 �C the

regulation of OsDREB1B gene expression is similar to what

happens at 10 �C, it is expected that after 2 h at 8 �C, thecDNA library will be enriched in OsDREB1B repressors

rather than activators.

This study showed that formation of heterodimers amongthe ZF-HD binding to the OsDREB1B promoter affected

their transactivation activity and that the dimerization

effect depends on the interacting partners. The repressor

activity of OsZHD1, which can form homodimers, was lost

when it was co-transformed with any of the other ZF-HD

TFs, with which it can form heterodimers. This suggests

that the heterodimers formed have different DNA-binding

specificities, which do not match the cis-elements present inthe promoter of OsDREB1B. Similarly, in mammals the

heterodimerization of C/EBPa with E2F proteins was also

shown to inhibit the transactivation activity of C/EBPaalone (Zaragoza et al., 2010). The dimerization of TFs has

long been described to regulate their binding to DNA and

also their transactivation activity. For instance, the TF

heterodimers Myc–Max and Mad–Max were shown to have

opposing functions in regulating transcription (Ayer et al.,1993). Moreover, homo- and heterodimers of two homeo-

domain proteins, Alx4 and Gcs, were described as having

different DNA-binding specificities, as well as transactivation

activities (Tucker and Wisdom, 1999).

This work allowed the identification of seven novel

players in the abiotic stress-signalling pathway. These novel

TFs bind to the promoter and interplay to repress the

expression of OsDREB1B, are involved in the response to

different abiotic stresses, and may also play a role in biotic

stress. Together with previous reports, these data suggestthat Zn-finger TFs may be a pivotal component in the

regulation of DREB1/CBF genes in plants.

Supplementary material

Supplementary data are available at JXB online.

Supplementary Fig. S1. Analysis of the OsDREB1B gene

expression under abiotic stress conditions.

Supplementary Fig. S2. Alignment of conserved DNA-

binding domains of the Zn-finger TFs.

Supplementary Fig. S3. Putative Zn-finger binding ele-

ments present in the OsDREB1B promoter.Supplementary Fig. S4. Expression analysis of the TF-

encoding genes under abiotic stress conditions.

Supplementary Fig. S5. Celullar localization of the Zn-

finger TFs.

Supplementary Table S1. Primers used to isolate the

OsDREB1B promoter fragments.

Supplementary Table S2. Primers used for semi-quantitative

RT-PCR.Supplementary Table S3. cis-Acting elements in the

promoter region of OsDREB1B.

Acknowledgements

We would like to thank Dr Alejandro Ferrando for

providing the BiFC vectors (www.ibmcp.upv.es/Ferrando-

LabVectors.php). DF (SFRH/BD/29258/2006), PB (SFRH/

BD/31594/2006), TS (SFRH/PD/31011/2006), TL (SFRH/

BPD/34943/2007), and SC (SFRH/BPD/64193/2009) are

grateful to Fundacao para a Ciencia e a Tecnologia (FCT)

for their fellowships. NS was supported by Programa

Ciencia 2007, financed by POPH (QREN). The researchwork was funded by FCT through the projects POCI/BIA-

BCM/56063/2004, PTDC/BIA_BCM/099836/2008, and

PEST-OE/EQB/LA0004/2011.

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