A cascade of transcription factor DREB2A and heat stresstranscription factor HsfA3 regulates the heat stressresponse of Arabidopsis

Franziska Schramm1, Jane Larkindale2, Elke Kiehlmann1, Arnab Ganguli1, Gisela Englich1, Elizabeth Vierling2 and

Pascal von Koskull-Doring1,*

1Institute of Molecular Biosciences, Biocenter N200/R306, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438

Frankfurt, Germany, and2Department of Biochemistry and Molecular Biophysics, University of Arizona, 1007 E. Lowell Street, Life Sciences South,

Tucson, AZ 85721, USA

Received 25 July 2007; revised 18 September 2007; accepted 26 September 2007.*For correspondence (fax +49 69 798 29286; e-mail doeringp@bio.uni-frankfurt.de).

Summary

The dehydration-responsive element binding protein (DREB)/C-repeat binding factor (CBF) family are the

classical transcriptional regulators involved in plant responses to drought, salt and cold stress. Recently it was

demonstrated that DREB2A is induced by heat stress (hs) and is a regulator of the hs response of Arabidopsis.

Here we provide molecular insights into the regulation and function of hs transcription factor HsfA3. Among

the 21 members of the Arabidopsis Hsf family, HsfA3 is the only Hsf that is transcriptionally induced during hs

by DREB2A, and HsfA3 in turn regulates the expression of Hsp-encoding genes. This transcription factor

cascade was reconstructed in transient GUS reporter assays in mesophyll protoplasts by showing that

DREB2A could activate the HsfA3 promoter, whereas HsfA3 in turn was shown to be a potent activator on the

promoters of Hsp genes. Direct binding to the corresponding promoters was demonstrated by electrophoretic

mobility shift assays, and the involvement of HsfA3 in the hs response in vivo was shown directly by

observation of reduced thermotolerance in HsfA3 mutant lines. Altogether these data demonstrate that HsfA3

is transcriptionally controlled by DREB2A and important for the establishment of thermotolerance.

Keywords: Arabidopsis, C-repeat binding proteins, DRE-binding proteins, heat stress response, heat stress

transcription factors, heat stress proteins.

Introduction

The development of complex abiotic stress signalling net-

works allowed terrestrial plants to grow and propagate under

the challenge of stressful conditions from which they cannot

escape, e.g. low or high temperatures, high salt or heavy

metal stress, or long-term water deficiency. In particular,

temperatures above the optimum are sensed as heat stress

(hs), which disturbs cellular protein homeostasis. The accu-

mulation of heat shock proteins (Hsps) is an essential part of

the hs response, and they are assumed to play a central role

in acquired thermotolerance in plants and other organisms

(Baniwal et al., 2004; Hartl and Hayer-Hartl, 2002; Haslbeck,

2002; Kotak et al., 2007b; Morimoto, 1998; Nakamoto and

Vıgh, 2007; Wang et al., 2004). The central regulators of

the expression of hs responsive genes are the heat stress

transcription factors (Hsfs) (Baniwal et al., 2004; Kotak et al.,

2007b; Miller and Mittler, 2006; Morimoto, 1998; Nover and

Scharf, 1997; Scharf et al., 1998b; Schoffl et al., 1998; Wu,

1995).

Sequencing of the Arabidopsis genome (The Arabidopsis

Genome Initiative, 2000) revealed a unique complexity of the

plant Hsf family, with 21 members belonging to three

classes: A, B and C (Nover et al., 2001). Other plant species

showed similar complexity: in the rice genome (Interna-

tional Rice Genome Sequencing Project, 2005) a minimum

of 23 Hsfs were identified (Baniwal et al., 2004; Xiong et al.,

2005), and for other plants a search of expressed sequence

tags (ESTs) databases indicated the presence of at least 18

Hsfs in tomato, and 34 Hsfs in soybean (Baniwal et al., 2004).

264 ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd

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From Arabidopsis, so far, Hsfs A1a, A1b, A2, A4a, A4c, A5, A9

and B1 have been functionally characterized in more detail

(Baniwal et al., 2007; Charng et al., 2007; Czarnecka-Verner

et al., 2000, 2004; Davletova et al., 2005; Hubel and Schoffl,

1994; Hubel et al., 1995; Kim and Schoffl, 2002; Kotak et al.,

2007a; Li et al., 2005; Lohmann et al., 2004; Nishizawa et al.,

2006; Panchuk et al., 2002; Prandl et al., 1998; Reindl and

Schoffl, 1998; Schramm et al., 2006; Wunderlich et al., 2003).

We could identify a single A3 type Hsf-encoding gene in

Arabidopsis, tomato and rice, forming together a clearly

separated branch in the phylogenetic tree of plant Hsfs

(Baniwal et al., 2004). Our functional analyses of the

C-terminal domains (CTDs) of the 21 Hsfs characterized the

CTD of HsfA3 as a potent activation domain. It was shown to

interact with the transcriptional machinery, and includes a

nuclear import signal (NLS) that is responsible for nuclear

localization (Kotak et al., 2004). Concerning the expression

of HsfA3 it was reported that its transcript level is elevated

under hs (Rizhsky et al., 2004). Strikingly, a recent study

identified that this hs-induced expression depends on the

dehydration-responsive element binding protein 2A (DRE-

B2A) (Sakuma et al., 2006b). Previously, DREB2A had only

been reported as an important transcription factor in the

drought and salt stress signalling pathways (Liu et al., 1998;

Nakashima et al., 2000; Sakuma et al., 2006a). DREB2A

belongs to a family of 14 AP2-domain transcription factors

(Sakuma et al., 2002), some members of which are also

referred to as C-repeat binding factors (CBFs). In contrast to

DREB2A and DREB2B, which were reported to predomi-

nantly play a role in the response to drought and salt stress,

the DREB1/CBF group (DREB1A/CBF3, DREB1B/CBF1 and

DREB1C/CBF2) play a central role as transcriptional regula-

tors of the cold stress response (Cook et al., 2004; Gilmour

et al., 1998; Shinozaki and Yamaguchi-Shinozaki, 2000;

Shinozaki et al., 2003; Stockinger et al., 1997; Thomashow,

1999). DREB1D/CBF4 is so far exceptional in that it is closely

related to the DREB1/CBF group, but is drought responsive

and not cold responsive (Haake et al., 2002).

The focus of this article is the functional analysis of

the hs-induced Arabidopsis HsfA3, with respect to its

hs-induced regulation by DREB2A and its role as a tran-

scriptional activator of Hsp-encoding genes. The data reveal

a novel pathway that regulates the hs response and

contributes to the development of thermotolerance.

Results

HsfA3 is expressed late in the hs response

Analysis of the transcript profiles of the 21 Arabidopsis Hsfs

during a time course of hs, as part of the AtGenExpress

project (http://www.arabidopsis.org/info/expression), indi-

cates that HsfA3 is not expressed under control conditions,

but that plants accumulate a significant level of HsfA3

transcripts after 3 h of hs, and that this level declines in early

recovery phase (Figure 1a). This pattern is in contrast to the

expression profiles of other hs-induced Hsfs (Hsfs A2, A7a,

A7b, B1, B2a and B2b), which already show higher transcript

levels after 0.5 h of hs, with a peak at 1 h and decline by 3 h

of hs. To determine if these changes in transcript accumu-

lation were reflected at the protein level, we raised poly-

clonal antibodies against the CTD of recombinantly

expressed HsfA3 (Kotak et al., 2004). Western blot analysis

with these antibodies demonstrates that the expression of

HsfA3 is strictly hs induced, and that this transcription factor

accumulates significantly as a protein during a later phase of

hs and in the early recovery phase after hs (Figure 1b). This is

true for essentially all tissues tested (seedlings in Figure 1b;

leaves, stem, flowers, green siliques and roots, data not

shown).

DREBs/CBFs selectively activate the HsfA3 promoter

in transient assays

A recent study determined that the hs-induced expression of

HsfA3 in Arabidopsis depends on DREB2A (Sakuma et al.,

2006b). DREB2A is itself hs induced in the early phase of hs

(Figure 1a). Sakuma et al. (2006b) demonstrated that in the

corresponding dreb2a knock-out line HsfA3 was no longer hs

inducible. The dependency of HsfA3 expression on DREB2A

was further supported by a P35S:DREB2A line that had in-

creased levels of HsfA3 under control conditions (Sakuma

et al., 2006b). Based on these results, we analysed the acti-

vation potential of the five best-characterized members of the

DREB/CBF family in a transient reporter assay in tobacco

mesophyll protoplasts, on a reporter construct containing

2-kb of upstream sequence from the HsfA3 promoter in

fusion to GUS (PHsfA3:GUS) (construct 1 in Figure 2a). The

results shown in Figure 2b demonstrate that DREB1/CBFs led

to only a moderate increase in GUS activity, but that DREB2A

and DREB2B led to a significant activation of the PHsfA3:GUS

reporter construct, showing up to a 20-fold increase in GUS

activity in comparison to the reporter alone. Remarkably,

none of the 21 Hsfs was able to induce the PHsfA3:GUS

reporter construct under these conditions, indicating that the

hs inducibility of HsfA3 depends strictly on DREB2A/B (Figure

S1), although many potential Hsf binding sites are predicted

in this promoter (Figure 2a; Nover et al., 2001). Again, this

indicates that it is not trivial to predict functional heat shock

element (HSE) clusters so far (see Schramm et al., 2006).

Furthermore, we tested similar 2-kb upstream sequences of

the promoters of the other 20 Hsf genes in fusion to GUS

(Figure S2), and the strong induction by DREB2A/2B was

exclusively observed for PHsfA3:GUS. Most PHsf:GUS reporter

constructs were not induced at all, with only PHsfA1e:GUS,

PHsfA6b:GUS and PHsfC1:GUS showing a moderate induction

by DREBs/CBFs (Figure S2). These data indicate that DREB2A

and DREB2B specifically regulate the HsfA3 promoter.

Role of Arabidopsis HsfA3 265

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DREB2A/2B bind directly to the HsfA3 promoter

Sequence analysis of the HsfA3 promoter led to the

prediction of three potential binding sites for DREBs/CBFs

(dehydration-responsive elements, DREs, are also referred

to as C-repeats, CRTs; Baker et al., 1994; Yamaguchi-Shino-

zaki and Shinozaki, 1994, 2005) (Figure 2a). We decided to

verify the functional significance of these binding sites

experimentally. To map functional binding sites in the pro-

moter of HsfA3, we generated a series of deletion constructs

of PHsfA3:GUS (Figure 2a) and tested them in transient

reporter assays (Figure 2b). Compared with the full-length

2-kb promoter fragment (construct 1), even a deletion of

1750 bp from the 5¢ end (construct 3) had only a minor

effect, indicating that this 250-bp construct containing DRE1

and DRE2, but lacking DRE3, is sufficient for the induction by

DREBs under these conditions. A mutation of DRE1 in this

minimal reporter construct led to a dramatic reduction in

activity (construct 4); furthermore, an additional mutation of

DRE2 led to a complete loss of GUS activity (construct 5),

thereby supporting a critical role for these elements.

To prove that DREBs bind directly to the mapped DRE

target sequences within the HsfA3 promoter, we performed

electrophoretic mobility shift assays (EMSAs) with multiple

PCR-amplified probes (templates were constructs 3–5 in

Figure 2a) and protein extracts from Escherichia coli

expressing different recombinant DREBs/CBFs (Figure 2c).

The results show that the probe with an intact DRE1

(amplified from construct 3 in Figure 2a) is also the most

important for this in vitro assay. DREB2A as well as DREB2B

bind with high affinity to this probe. In addition, DREB1B/

CBF1 binds weakly to this probe, but DREB1A/CBF3,

DREB1C/CBF2, HsfA2 or GST alone do not (Figure 2c). EMSA

with the corresponding probe carrying a mutated DRE1 led

to a strong reduction in affinity, and mutation of both DRE1

and DRE2 led to a complete loss of binding (Figure 2c,

probes amplified from constructs 4 and 5, respectively,

shown in Figure 2a). This correlates nicely with the results

obtained in protoplasts (Figure 2b). Mutation of DRE2 alone

showed only some reduction for DREB2A and DREB2B, but

not for DREB1B/CBF1, in both test systems (data not shown).

The specificity of the EMSA is further supported by the use

of a probe from the RD29A (also referred to as COR78/LTI78)

promoter, which shows binding of all DREBs/CBFs as

previously described (Sakuma et al., 2002, 2006a). In addi-

tion, a probe from the Hsp17.4-CI promoter was only bound

by HsfA2, as previously reported (Schramm et al., 2006).

Analysis of potential target promoters of

DREB2A/B and HsfA3

Similar to HsfA3, DREB2A-dependent expression has been

reported for genes encoding Hsps (e.g. Hsp18.1-CI, Hsp26.5-

MII), in addition to well-known DREB2A target genes, such

Figure 1. Expression profiles of heat stress (hs)

heat stress transcription factor (Hsf) and dehy-

dration-responsive element binding (DREB)/

C-repeat binding factor (CBF) genes under hs.

(a) Normalized and averaged signal intensities

were visualized as ‘heat maps’, with retrans-

formed linear signal intensities from the AtGen-

Express hs series, for the Hsf family and for the

best-characterized members from the DREB/

CBF family (see text for references). Transcript

levels for Actin7 (Act7) and Ubiquitin11 (Ubq11)

are shown as controls. The corresponding col-

our bar for the signal intensities of the transcript

levels is shown below. For a description of

the selected samples and further details, see

Experimental procedures and http://www.

arabidopsis.org/.

(b) Immunoblot analysis of protein extracts

from 7-day-old Arabidopsis seedlings before

hs as a control (C, 0), during hs (Hs, 1 h and 3 h)

and in the recovery phase after hs (Rec, +1 h and

+3 h). Heat stress was performed at 36�C. For

further details, see Experimental procedures.

266 Franziska Schramm et al.

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 264–274

as COR47/RD17, RD29A and GolS1 (Sakuma et al., 2006b).

As target genes of HsfA3 and the contribution of HsfA3 to

thermotolerance have not been reported, we wanted to

determine whether DREB2A activates Hsp genes directly or

via HsfA3. Similar to experiments shown in Figure 2b, we

analyzed the activation potential of the five DREB/CBF

constructs and an HsfA3 expression construct on various

potential target promoters in fusion to GUS (Figure S3). We

could classify the results in three groups: (i) the reporter

constructs for four Hsp genes, represented by PHsp18.1-CI:-

GUS, showed no induction by DREBs/CBFs, but a specific

induction by HsfA3 (Figure S3A); (ii) reporter constructs

represented by PHsp26.5-MII

:GUS, including two other Hsp genes

and the GolS1 gene, showed minor induction by DREBs/

Figure 2. DREB2A/2B binding to the HsfA3 promoter in transient GUS reporter assays and electrophoretic mobility shift assays (EMSAs).

(a) Schematic representation of the HsfA3 promoter fragments that were fused to GUS and used in the corresponding transient reporter assay performed in (b).

Predicted dehydration-responsive elements (DREs) as potential binding sites for DRE binding proteins (DREBs) are indicated (see text for references). Heat stress

elements (HSE) are drawn according to the nomenclature of Nover et al. (2001). An open box above the line represents an active HSE head module: a(g,t,c)GAAn,

a(g,t,c)GnAn or a(g,t,c)GAnn. Numbers in between indicate the distance in bp. The corresponding filled box represents an inactive head module with five

nucleotides, but lacking the invariant G and/or both A residues. An open box below the line represents an active HSE tail module: nTTCt(a,g,c), nTnCt(a,g,c) or

nnTCt(a,g,c). The corresponding filled box represents an inactive tail module with five nucleotides, but lacking the invariant c and/or both T residues. X represents a

mutated form of the DRE sites. The region amplified from constructs 3–5 for the corresponding EMSA shown in (c) is also indicated (marked as probe).

(b) The activator potential of P35S:DREB constructs was tested in tobacco protoplasts on reporter constructs containing different deleted forms of the HsfA3 promoter

fused to GUS, as illustrated in (a). The resulting GUS activities (relative fluorescence units, RFU) are presented � co-transformation with the P35S:DREB expression

constructs indicated. Error bars correspond to the SD of three independent replicates. For details see Experimental procedures and Table S1.

(c) The corresponding EMSA with different PCR-amplified and radioactively labelled fragments of the HsfA3 promoter region from )112 to )269 bp, with reference

to the start codon. The corresponding templates were constructs 3–5 shown in (a) containing a mutation in DRE1 (HsfA3mDRE1) or in both DRE1 and DRE2

(HsfA3mDRE1+2). As controls, promoter regions from RD29A and Hsp17.4-CI were analyzed similarly. The electrophoretic mobility of the probe after incubation with

Escherichia coli protein extracts recombinantly expressing GST, GST-DREB1A(CBF3), GST-DREB1B(CBF1), GST-DREB1C(CBF2), GST-DREB2A, GST-DREB2B or

GST-HsfA2 are shown. Note that all fragments were about 170 bp in length. For details see Experimental procedures and Table S2.

Role of Arabidopsis HsfA3 267

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CBFs, in addition to a strong induction by HsfA3 (Figure

S3B); and (iii) reporter constructs of three described DREB/

CBF-regulated genes, represented by PRD29A:GUS, showed a

strong induction by DREBs/CBFs, as previously reported (Liu

et al., 1998; Sakuma et al., 2006a; Yamaguchi-Shinozaki and

Shinozaki, 1994), but no induction by HsfA3 (Figure S3C).

These results clearly indicate Hsp genes reported to be

DREB2A dependent are actually activated dominantly or

even exclusively via HsfA3.

In order to reconstruct this transcription cascade in a

single cell, we used a transient dual activator–reporter GUS

system in tobacco mesophyll protoplasts, as established

previously (Kotak et al., 2007a). We cloned HsfA3-3HA under

the control of its own hs-inducible promoter in a plant

expression vector (PHsfA3:HsfA3-3HA), and then analyzed its

activator potential either alone or in combination with

DREB2A on reporter constructs containing 1-kb upstream

sequences of selected Hsp promoters in fusion to GUS

(PHsp:GUS, see Experimental procedures for details). As

shown in Figure 3, co-transformation of PHsfA3:HsfA3-3HA

and the PHsp:GUS reporter constructs showed no GUS

activity and no detectable HsfA3-3HA protein. However,

the presence of DREB2A led to the expression of HsfA3-3HA

from the PHsfA3:HsfA3-3HA construct along with a 10–15-fold

increase in GUS activity expressed from the co-transformed

PHsp:GUS constructs (Figure 3). Furthermore, in the absence

of PHsfA3:HsfA3-3HA, DREB2A showed no induction of the

Hsp18.1-CI promoter driven GUS reporter, but induced the

PHsp26.5-MII:GUS to a minor extent. Again, as a control, the

PRD29A:GUS construct was shown to be induced as expected

by DREB2A, but not by HsfA3 (Figure 3). In this context it is

worth remarking that DREB2B showed very similar results to

DREB2A (data not shown). Accordingly, the data obtained

from the transient dual activator–reporter GUS system

strongly suggests a transcriptional cascade, in which

DREB2A regulates the expression of HsfA3 as an essential

transcription factor for the expression of many Hsp genes

(Figure 3).

Analysis of HsfA3 binding to potential target promoters

To analyze whether HsfA3 and DREBs/CBFs bind directly to

the promoter sequences of Hsp18.1-CI and Hsp26.5-MII, we

performed EMSAs with different PCR-amplified probes and

protein extracts from E. coli expressing recombinant HsfA3

and DREBs/CBFs, similar to the experiments shown in Figure

2(c) (Figure 4). We used different PCR-amplified probes of

approximately 170 bp each to map the binding of

recombinant HsfA3 and DREBs/CBFs to approximately 500-

bp promoter sequences of the Hsp18.1-CI and Hsp26.5-MII

genes (Figure 4a/c). The results clearly demonstrate that

only HsfA3 binds with high affinity to a TATA-proximal HSE-

containing promoter fragment of the Hsp18.1-CI gene (pro-

be C in Figure 4b). We analyzed up to 1000 bp, but did not

observe any binding of DREBs/CBFs to Hsp18.1-CI promoter

fragments (data not shown). In the case of the Hsp26.5-MII

promoter we observed a similar binding of HsfA3 to a TATA-

proximal HSE-containing promoter region (probe C in

Figure 4d), but in addition we detected weak binding of

DREB2A and DREB2B to a different site (probe B in Figure

4d), which contains a predicted DREB binding site (DRE in

Figure 4c). The specificity of the EMSA is further supported

by the use of a mutant form of HsfA3 with an R109A ex-

change in the DBD, which shows no binding potential

(HsfA3m samples).

Impact of HsfA3 on the hs response

Overexpression of DREB2A led to the induction of hs-related

genes, including HsfA3, Hsp18.1-CI and Hsp26.5-MII

(Sakuma et al., 2006b). This was accompanied by higher

tolerance to hs treatments, whereas DREB2A knock-out

mutants showed reduced HsfA3 and Hsp transcript levels

together with a reduced thermotolerance. So far the target

genes of HsfA3 and the contribution of HsfA3 to thermotol-

erance have not been reported. Therefore, we analyzed two

plant lines that did not express HsfA3 at detectable levels,

one a SALK T-DNA insertion mutant (hsfA3) and the other an

RNAi line (hsfA3 RNAi) (Figure 5d), in different thermotol-

erance assays, as established by Larkindale et al. (2005)

(Figure 5a–c). The hot1-3 mutant was used as a positive

Figure 3. Dual activator–reporter assay in mesophyll protoplasts.

The activator potential of HsfA3 and DREB2A were tested in tobacco

mesophyll protoplasts by transient co-transformation of expression con-

structs for DREB2A or HsfA3-3HA under control of the constitutive 35SCaMV

promoter (P35S:DREB2A, P35S:HsfA3-3HA) or, in the case of PHsfA3:HsfA3-3HA,

under control of its own inducible promoter with reporter constructs

containing 1-kb promoter fragments of Hsp18.1-CI, Hsp26.5-MII or RD29A

fused to GUS (for details see Experimental procedures and Table S1), as

indicated above the corresponding samples. The resulting GUS activities

(relative fluorescence units, RFU) are presented with error bars representing

the SD of three independent replicates. As shown below, expression of the

HsfA3-3HA protein was monitored by immunoblot analysis of the corre-

sponding samples using an antibody against the 3HA-tag (a HA).

268 Franziska Schramm et al.

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 264–274

control, as this plant is null for Hsp101 and has previously

been shown to be defective in thermotolerance (Hong and

Vierling, 2001; Larkindale et al., 2005). In an assay for basal

thermotolerance of germination, both lines (hsfA3 and

hsfA3 RNAi) showed a 30–40% reduced rate of germination

followed by the development of green cotyledons after

220 min of hs, compared with wild type (wt) (Figure 5a). In

an assay for acquired thermotolerance tested by measuring

elongation of dark-grown 2.5 day-old seedlings, the means

of hypocotyl elongation of both HsfA3 mutant lines was

reduced to 40–50% compared with wt under all hs conditions

tested (Figure 5b). Finally, a survival assay of 7-day-old

seedlings after hs showed a reduced survival rate of

approximately 60% compared with wt for both HsfA3 mutant

lines (Figure 5c). This reduced thermotolerance at different

developmental stages correlated with the observation that

both HsfA3 mutant lines have reduced levels of Hsp101, as

well as sHsps, under hs (Figure 5d).

Discussion

A novel transcriptional cascade containing HsfA3 and DREBs

The data presented here demonstrate that HsfA3 is excep-

tional among the 21 members of the Arabidopsis Hsf family,

being hs induced but regulated by DREB2A and not by Hsfs.

Recently, it was reported that the expression of HsfA3,

Hsp18.1-CI, Hsp26.5-MII and other transcripts was depen-

dent on the presence of DREB2A, shown through evidence

from transgenic plants with altered DREB2A levels (knock-

out versus overexpression) (Sakuma et al., 2006b). Similar

results were obtained by overexpression of the homologous

protein ZmDREB2A from Zea mays in Arabidopsis (Qin

et al., 2007). Using transient GUS reporter assays, as well as

EMSAs, we have shown that DREB2A and DREB2B, and to a

minor extent DREB1B/CBF1, are able to bind specifically to

the HsfA3 promoter, with the most important binding site

determined to be DRE1 with the sequence AACCGACAA

(Figure 2). This sequence includes the well-known core se-

quence CCGAC of the DRE/ CRT, described as a cis-acting

element for the binding of DREBs/CBFs (Baker et al., 1994;

Liu et al., 1998; Maruyama et al., 2004; Sakuma et al., 2002;

Stockinger et al., 1997; Thomashow, 1999; Yamaguchi-

Shinozaki and Shinozaki, 1994, 2005). In our experiments

DREB2B seems to be functionally similar to DREB2A, but

there is no evidence from in planta analyses for a similar

function in the hs response, particularly in the regulation of

HsfA3 expression. We have analyzed a DREB2B knock-out

line, but it showed neither a defect in HsfA3 expression nor a

defect in thermotolerance (data not shown). This observa-

tion might be explained by the very low expression level of

DREB2B compared with DREB2A (Figure 1). In this respect it is

Figure 4. Binding of HsfA3 and dehydration-responsive element binding proteins (DREBs) on promoter fragments of potential target genes in electrophoretic

mobility shift assays (EMSAs).

(a, c) Promoter structure of Hsp18.1-CI and Hsp26.5-MII. Heat shock elements (HSEs), the 5¢ untranslated region (5¢-UTR) and the transcriptional start site are

indicated. For a detailed description of HSEs see also Figure 2(a). PCR-amplified regions used in the EMSAs are shown in (B) and (D) respectively. Note that all

fragments were about 170 bp in length (see Table S2).

(b, d) The corresponding EMSA with the different PCR-amplified and radioactively labelled fragments of the promoter sequences as indicated in (A) for Hsp18.1-CI

and in (c) for Hsp26.5-MII. The electrophoretic mobility of the probe is shown after incubation with Escherichia coli protein extracts recombinantly expressing GST,

GST-DREB1A(CBF3), GST-DREB1B(CBF1), GST-DREB1C(CBF2), GST-DREB2A, GST-DREB2B, GST-HsfA3 or GST-HsfA3m with an R98A exchange in the DNA-binding

domain. Note that all fragments were about 170 bp in length. For details see Experimental procedures and Table S2. For the control of the binding of DREBs on the

RD29A promoter, see Figure 2(c).

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ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 264–274

also intriguing to speculate about a potential DREB-depen-

dent expression of HsfA3 under cold and salt stress, as indi-

cated from its expression profile in the AtGenExpress stress

series and a specific function in these pathways (Figure S4).

The target genes of HsfA3 and its role in the hs response

have not previously been reported. Transient GUS reporter

assays presented here indicate that HsfA3, but not DREB2A,

was able to activate strongly representative hs-inducible

promoters (Hsp18.1-CI and others), whereas Hsp26.5-MIIwas modestly induced by DREB2A/B in addition to HsfA3

(Figure 3 and Figure S3). The requirement for HsfA3 was

further supported by EMSAs, demonstrating that HsfA3

binds to a TATA-proximal HSE cluster with high affinity,

whereas no DREB protein was found to bind to any

sequence within 1 kb of this promoter (Figure 4b). These

results indicate that Hsp18.1-CI is not co-regulated with

HsfA3 by DREB2A, as suggested by microarray analyses of

plants with altered DREB2A levels (Sakuma et al., 2006b),

but rather that it is regulated by the transcriptional cascade

DREB2A fi HsfA3 fi Hsp18.1-CI. To support this argu-

ment we reconstructed this regulatory cascade in a dual

activator–reporter assay in mesophyll protoplasts, where

only co-transformation of P35S:DREB2A fi PHsfA3:HsfA3-

3HA fi PHsp18.1-CI:GUS led to high GUS activities, together

with detectable HsfA3 accumulation (Figure 3). This was also

reconstituted with other Hsp promoters (Figure 3, Figure S3

and data not shown). Our analyses of two HsfA3 null lines

(hsfA3 and hsfA3 RNAi) indicate that the expression of sHsps

as well as Hsp101 is significantly reduced under hs in both

mutant lines (Figure 5d). Finally, we analysed the impact of

HsfA3 on thermotolerance. Under all assay conditions, both

HsfA3 null lines showed a reduced thermotolerance of

40–60%, indicating that it is an essential factor for Hsp

expression and thermotolerance.

Role of HsfA3 in the hs network of Hsfs

Previously, Hsfs A1a, A1b and A2 have been reported to

modulate the hs response in Arabidopsis (Busch et al., 2005;

Charng et al., 2007; Lohmann et al., 2004; Nishizawa et al.,

2006; Schramm et al., 2006). Analysis of the double knock-

out of the HsfA1a and HsfA1b genes (Busch et al., 2005)

indicates that HsfA1a and/or HsfA1b are responsible for the

hs-induced expression of a subset of genes encoding sHsps,

Hsp70, Hsp101, Hsfs (B1, B2b and A7a) as well as enzymes of

sugar metabolism, e.g. inositol-3-phosphate-synthase (IPS2)

and galactinol synthase (GolS1). So far, no master regulator

of the hs response with severe defects in thermotolerance,

as seen with a co-suppression line of tomato HsfA1a (Mishra

et al., 2002), could be identified in Arabidopsis. In contrast to

the constitutively expressed Hsfs A1a and A1b, HsfA2

accumulates as a strictly hs-induced protein in practically all

tissues (Schramm et al., 2006). Analysis of the correspond-

ing knock-out line led to the identification of major target

genes, including ascorbate peroxidase 2 (APX2), several

sHsps, and individual isoforms of the Hsp70 and Hsp101

families (Schramm et al., 2006). HsfA2 knock-out plants

exhibited thermotolerance defects after repeated hs

Figure 5. Phenotypes in mesophyll protoplasts. of HsfA3 mutant lines.

(a–c) Phenotypic assays for heat tolerance of mutant and wild-type (Wt) plants. Results shown are an average of three independent experiments; error bars represent

SD. Black bars, Col-0 (Wt); white bars, hot1-3; light-grey bars, hsfa3 insertion mutant; mid-grey bars, hsfa3 RNAi line.

(a) Percentage of seeds germinated 3 days after heating to 45�C without pre-treatment for 220 min.

(b) Hypocotyl elongation of 2.5-day-old seedlings after treatment at 38�C for 90 min, 22�C for 120 min and 45�C for 180 or 200 min, after 2.5 days of recovery.

(c) Survival of 7-day-old plants heated to 38�C for 90 min, 22�C for 120 min and 45�C for 120, 140 or 160 min, after 5 days of recovery.

(d) Immunoblots monitoring protein accumulation of HsfA3, Hsp101, sHsp CI and sHsp CII for the wild-type and mutant plants. Proteins were extracted from 7-day-

old plants heated to 38�C for 90 min, followed by 3 h at 22�C. For further details, see Experimental procedures.

270 Franziska Schramm et al.

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 264–274

treatments with extended recovery phases (Charng et al.,

2007). This is in agreement with another study analyzing this

knock-out line under high-light stress, and with observations

from unstressed leaves of an HsfA2-overexpression line

(Nishizawa et al., 2006). Taken together, these studies sug-

gest a regulatory amplifier role of HsfA2 for the expression

of a subset of genes under hs. Surprisingly, the expression

of most HsfA2-regulated sHsp genes seem also to be

dependent on HsfA1a/A1b (Busch et al., 2005), indicating

cooperation between HsfA1a/A1b and HsfA2. This com-

plexity of the Hsf network in regulating the expression of Hsp

genes is increased even further by our preliminary results,

which indicate that HsfA3 is able to interact specifically with

Hsfs from the HsfA1 group as well as with HsfA2 (data not

shown). Interaction of HsfA3 and HsfA1 is also supported by

yeast two-hybrid interaction of tomato HsfA3 and HsfA1a

(Bharti et al., 2000). Thus, these results suggest the model

summarized in Figure 6. The immediate early hs response

seems to be regulated by the constitutively expressed

HsfA1a/A1b (Lohmann et al., 2004), which is supported and

amplified by cooperation with the hs-induced HsfA2

(Nishizawa et al., 2006; Schramm et al., 2006). Then, for a

later phase of the hs response, it is tempting to speculate

that the delayed hs-induced HsfA3 might be an essential

additional factor in this network to establish full thermotol-

erance, possibly by forming hetero-oligomeric complexes

with Hsfs A1a, A1b and A2 (Figure 6). In this respect it is

worth noting that we have preliminary results from transient

reporter assays supporting this concept of Hsf superactiva-

tor complexes on Hsp promoters (data not shown), although

the nature of their composition is still unknown.

Functional roles of Arabidopsis Hsfs beyond

the hs response

So far each analysis of an Hsf mutant line has led to the

identification of a specific pathway. In addition to the

examples mentioned already, which represent essential

parts for regulation of the hs response, there are further

examples indicating that Hsfs seem to be involved in sig-

nalling pathways other than the hs response. Analysis of a

knock-out line of the constitutively expressed ascorbate

peroxidase 1 (APX1) led to an upregulation of HsfA4a, and

in turn the overexpression of a dominant negative form

of HsfA4a led to a decrease in ZAT12 and APX1 levels

(Davletova et al., 2005). This suggests that HsfA4a might

play a role as a sensor for oxygen radicals (Miller and Mittler,

2006). HsfA4 activity was shown to be controlled by hete-

rodimerization with HsfA5 as the selective repressor (Bani-

wal et al., 2007). Within the family of 21 Hsfs, HsfA9 is so far

exceptional, in that it is exclusively expressed in the late

stages of seed development and is not induced by any

stress. This exclusive developmental expression of HsfA9

depends on the seed-specific transcription factor ABSCISIC

ACID–INSENSITIVE3 (ABI3), and HsfA9 in turn is the essen-

tial factor for the developmental expression of Hsp genes

during the late seed maturation phase (Kotak et al., 2007a).

Although detailed functional analyses of Hsfs with specific

expression patterns are limited to the examples described

above, the expression profiles of the 21 Hsfs extracted from

the AtGenExpress microarray data (Figure S4; Kotak et al.,

2007a) indicate that many more Hsfs might have specialized

functions integrated into different signalling pathways in

response to abiotic or biotic stress, or as part of develop-

mental programs. Together with other transcription factors

these form a complex regulatory network, essential for

plants to resist rapidly changing environmental stresses and

to propagate successfully under these conditions.

Experimental procedures

Plant material and hs conditions

The Arabidopsis line SALK_011107 containing a T-DNA insertion inthe HsfA3 gene (hsfA3) was used for characterization of HsfA3during hs in planta. The T-DNA integration site was verified, andhomozygous plants were identified and used for further character-ization. Furthermore, we analyzed an independent HsfA3 RNAi line(hsfA3 RNAi) expressing an inverted repeat of a fragment encodingthe CTD (cloned as a SalI/NotI fragment described in Kotak et al.,

Figure 6. Model for the regulation of the heat stress (hs) response in

Arabidopsis.

The immediate early hs response seems to be regulated by the constitutively

expressed hs transcription factors (Hsfs) A1a/A1b by a yet unknown triggering

signal (Busch et al., 2005; Lohmann et al., 2004). In contrast to this, HsfA2

accumulates rapidly after the onset of hs, whereas its transcriptional

regulation is not known (Charng et al., 2007; Nishizawa et al., 2006; Schramm

et al., 2006). The overlapping set of its target genes indicates cooperation with

HsfA1a/A1b on hs-induced Hsp promoters (Busch et al., 2005; Nishizawa

et al., 2006; Schramm et al., 2006). DREB2A is another hs-induced transcrip-

tion factor, which leads to a delayed hs-induced expression of HsfA3 (Sakuma

et al., 2006b). Finally, HsfA3 might be an essential additional factor in this

network regulating the expression of Hsp genes by functional cooperation

with Hsfs A1a/A1b/A2 to establish full thermotolerance. See text for further

details and references.

Role of Arabidopsis HsfA3 271

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 264–274

2004 in pJawohl8, a vector kindly provided by Drs Imre E.Somssichand Bekir Ulker, Max Planck Institute for Plant Breeding Research,Cologne, Germany) line, supporting our main conclusions. As acontrol for thermotolerance assays, the well-characterizedhot1-3 null mutant was used (Hong and Vierling, 2001).Thermotolerance assays were performed as described byLarkindale et al. (2005).

General materials and experimental procedures

Standard procedures were used for gene technology work. Forcloning, PCR was performed using the Taq Plus Precision System(Stratagene, http://www.stratagene.com). DNA fragments werepurified by ‘QIAquick gel extraction kit’ (Qiagen, http://www.qiagen.com). GUS reporter assays and analysis of protein expres-sion in tobacco protoplasts were described previously (Doring et al.,2000; Kotak et al., 2004; Scharf et al., 1998a). Protein extraction andanalysis from Arabidopsis tissues were performed as described fortomato (Mishra et al., 2002). For Western blot analysis of HsfA3, aGST-tagged C-terminal fragment (aa 149–412, see Kotak et al.,2004) expressed in E. coli and purified on GST-Sepharose (Amer-sham, http://www.amersham.com) was used for immunization ofguinea pigs (Eurogentec, http://www.eurogentec.com). The antiseraagainst-Hsp17-CI/CII and Hsp101 were described previously (Hongand Vierling, 2001; Wehmeyer et al., 1996). Secondary antibodiesagainst guinea pig, mouse or rabbit immunoglobulins conjugatedwith horseradish peroxidase were obtained from Sigma-Aldrich(http://www.sigmaaldrich.com).

Activator and reporter constructs for plant cells

Plant expression vectors used are based on the pRT series of vectors(Doring et al., 2000; Topfer et al., 1988). For expression of HsfA3 andDREBs in mesophyll protoplasts, the coding sequence was PCRamplified from cDNA of heat stressed cell cultures with gene-spe-cific primers (see Table S1) introducing 5¢ KpnI and 3¢ NotI sites, andcloned into pRT103 (Topfer et al., 1988). For functional analysis ofpromoters, a maximum of 2 kb of the sequence upstream of thestart codon was PCR amplified from genomic DNA with gene spe-cific primers introducing 5¢ PstI and 3¢ NcoI or XhoI sites, andinserted in fusion to the coding frame of the GUS gene in pBT2gus(Topfer et al., 1988). For 5¢ deletion constructs of the HsfA3promoter, corresponding sequence-specific primers were used.Mutations of DRE1 and DRE2 were generated by PCR-based muta-genesis, as described previously (Lyck et al., 1997), introducing anXhoI or BamHI site instead of the original sequence. Details, primersand constructs are given in Table S1.

Protein expression in E. coli and EMSAs

To analyze HsfA3 and DREBs in EMSAs GST fusion proteins weregenerated. The pGST-HsfA2 expression construct described inSchramm et al. (2006) was cut with NcoI/NotI, and the insertreplaced by the 3HA-HsfA3 open reading frame (ORF) or 3HA-DREBORF containing fragment excised with NcoI/NotI from the pRT-Hsfor pRT-DREB constructs described above. The corresponding DBDmutant form with an R109A exchange was generated by PCR-basedmutagenesis, as described previously (Lyck et al., 1997), using theprimer 2850R: ccatatgtgttcagctgtgcgacgaagctgg.

Expression of recombinant proteins in E. coli strain BL21RIL(Stratagene), labelling of PCR probes with 32P (gene-specific primerin Table S2) and performance of EMSAs were described inSchramm et al. (2006). X-ray films were exposed for 1–3 h.

Microarray analysis of expression data

For expression profiles of selected genes from the AtGenExpressmicroarray database, the signal intensities were gcRMA normalizedand averaged (available at http://www.weigelworld.org/resources/microarray/AtGenExpress), and visualized as ‘heat maps’ (withGENESPRING Version 7.2) with retransformed linear signal intensi-ties. For description of the samples and further details, see http://www.arabidopsis.org/ and http://www.weigelworld.org/resources/microarray/AtGenExpress.

Acknowledgements

We thank Sachin Kotak for critical discussion during the preparationof the manuscript, and Dr Stefan Henz for providing the averagedgcRMA data set. This work was supported by grants from theDeutsche Forschungsgemeinschaft (AFGN grant KO2888/1-1/2 toPvK-D) and the United States Department of Agriculture (USDA-NRICGP 3510014857 to EV).

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Activity of Hsfs and dehydration-responsive elementbinding proteins (DREBs) on the HsfA3 promoter in transient GUSreporter assays.Figure S2. Activity of dehydration-responsive element bindingproteins (DREBs) on Hsf promoters in transient GUS reporterassays.Figure S3. Activity of HsfA3 and dehydration-responsive elementbinding proteins (DREBs) on potential target promoters in transientGUS reporter assays.Figure S4. Expression profiles of Hsf and dehydration-responsiveelement binding (DREB)/C-repeat binding factor (CBF) genes in theAtGenExpress stress series.Table S1. Details for construction of the activator and reporterconstructs.Table S2. Details for the probes used for electrophoretic mobilityshift assays (EMSA).This material is available as part of the online article from http://www.blackwell-synergy.comPlease note: Blackwell Publishing are not responsible for the contentor functionality of any supplementary materials supplied by theauthors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

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