orthologs of the class a4 heat shock transcription …...2009/12/22 · orthologs of the class a4...

Orthologs of the Class A4 Heat Shock Transcription FactorHsfA4a Confer Cadmium Tolerance in Wheat and Rice C W

Donghwan Shim,a Jae-Ung Hwang,a Joohyun Lee,a,1 Sichul Lee,b Yunjung Choi,a Gynheung An,b

Enrico Martinoia,a,c and Youngsook Leea,2

a POSTECH-UZH Global Research Laboratory, Division of Integrative Biology and Biotechnology, Pohang University of Science

and Technology, Pohang, 790-784, Koreab Division of Molecular and Life Science, Biotechnology Research Center, Pohang University of Science and Technology,

Pohang 790-784, Koreac Institute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland

Cadmium (Cd) is a widespread soil pollutant; thus, the underlying molecular controls of plant Cd tolerance are of substantial

interest. A screen for wheat (Triticum aestivum) genes that confer Cd tolerance to a Cd hypersensitive yeast strain identified

Heat shock transcription factor A4a (HsfA4a). Ta HsfA4a is most similar to the class A4 Hsfs from monocots. The most

closely related rice (Oryza sativa) homolog, Os HsfA4a, conferred Cd tolerance in yeast, as did Ta HsfA4a, but the second

most closely related rice homolog, Os HsfA4d, did not. Cd tolerance was enhanced in rice plants expressing Ta HsfA4a and

decreased in rice plants with knocked-down expression of Os HsfA4a. An analysis of the functional domain using chimeric

proteins constructed from Ta HsfA4a and Os HsfA4d revealed that the DNA binding domain (DBD) of HsfA4a is critical for Cd

tolerance, and within the DBD, Ala-31 and Leu-42 are important for Cd tolerance. Moreover, Ta HsfA4a–mediated Cd

resistance in yeast requires metallothionein (MT). In the roots of wheat and rice, Cd stress caused increases in HsfA4a

expression, together the MT genes. Our findings thus suggest that HsfA4a of wheat and rice confers Cd tolerance by

upregulating MT gene expression in planta.

INTRODUCTION

Cadmium (Cd) is one of the most dangerous heavy metals

present in the environment and poses a significant risk to human

health (Jarup et al., 1998). Cd inactivates or denatures proteins

by binding to sulfhydryl groups, which causes cellular damageby

displacing cofactors from a variety of proteins, including tran-

scription factors and enzymes (Goyer, 1997; Schutzendubel

et al., 2001). In addition, Cd induces oxidative stress, which in

turn mediates cellular damage in many plants and animals (Hart

et al., 1999; Sandalio et al., 2001).

Several mechanisms of defense against Cd-induced damage

have been elucidated in plants and other organisms. The most

frequently used are chelation, extrusion, sequestration of Cd,

and dissipation of the reactive oxygen species (ROS) generated

by Cd (Clemens et al., 2002). Cys-rich proteins are often used for

chelation of heavy metals. The small, Cys-rich metallothionein

proteins are the main chelators of Cd and other heavy metals in

animals (including humans) (Imura et al., 1989; Clemens, 2001).

Bakers’ yeast contains a single metallothionein gene, CUP1

(copper resistance associated protein), which sharesmany of the

basic features of the animal metallothionein genes (Butt and

Ecker, 1987). Metallothioneins also play a role in heavy metal

chelation and heavy metal homeostasis in plants (Zhou and

Goldsbrough, 1994). In addition to metallothioneins, plants syn-

thesize the glutathione-derived phytochelatins, which bind Cd

and thereby reduce the interaction of its free ionic form with

important cytosolic proteins (Clemens et al., 1999; Ha et al., 1999).

Extrusion of Cd by the ABC transporter PDR8 (pleiotropic drug

resistance 8) at the plasma membrane also contributes to Cd

tolerance in plants (Kim et al., 2007). A P-type ATPase in

Escherichia coli (ZntA) also pumps Cd out at the cell membrane

(Lee et al., 2003), thereby contributing to Cd tolerance. Seques-

tration of heavy metals is often performed by ATP binding

cassette (ABC) transporters localized to the vacuolar membrane.

YCF1 (yeast Cd factor 1) is an ABC transporter of Saccharomy-

ces cerevisiae that contributes to Cd resistance by pumping

glutathione-conjugated Cd into the vacuole (Li et al., 1997).

HMT1 (heavy metal transporter 1), which is a half-size ABC

transporter from Schizosaccharomyces pombe, also transports

phytochelatin-Cd and glutathione-Cd complexes into the vacu-

ole (Ortiz et al., 1995; Preveral et al., 2009). ROS dissipation is

mainly achieved by superoxide dismutases localized in the

cytosol, mitochondria, and chloroplasts (Alscher et al., 2002).

Anti-oxidant/reducing agents, which include glutathione and

ascorbate, also protect cells from the ROS generated by Cd.

These diverse mechanisms of detoxification can be controlled

by a single transcription factor that modulates the expression of

1Current address: Department of Biochemistry, University of Wisconsin,Madison, WI 53706.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Youngsook Lee([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.066902

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

reduces the time to publication by several weeks.

The Plant Cell Preview, www.aspb.org ã 2009 American Society of Plant Biologists 1 of 13

multiple proteins, which use different and specific detoxification

mechanisms. For example, in animal systems, the metal-

responsive transcription factor 1 (MTF-1) is induced by Cd, Cu,

and H2O2 (Dalton et al., 2000); in turn, MTF-1 upregulates me-

tallothionein (Zhang et al., 2003), the ABC transporter CG10505

(which is suggested to sequester Cd into the vacuole, similarly to

YCF1) (Yepiskoposyan et al., 2006), and the Zn efflux transporter

ZnT-1 (Langmade et al., 2000), thus providing multiple ways to

protect cells from heavy metal–induced damage. MTF-1 is con-

served from arthropods to mammals; however, it has not been

described in plants. Importantly, the transcription factors that

regulate the mechanisms of Cd detoxification in plants have

been poorly studied.

The heat shock transcription factors (Hsfs) of animals and fungi

play central roles in protecting cells from damage caused by

various stress conditions, including heat, infection, inflammation,

and pharmacological agents, via the activation of gene expres-

sion (Chen and Parker, 2002). The overall basic structure of Hsfs

and their consensus DNA binding site (i.e., the heat shock

element) are conserved from yeast to humans (Wu, 1995). Yeast

and humans have only limited numbers of Hsfs (one in yeast and

three in humans; Nakai, 1999). By contrast, plants possess large

families of genes encoding Hsfs; for example, Arabidopsis

thaliana and rice (Oryza sativa) have 21 and 23 Hsf genes,

respectively (Nover et al., 2001; von Koskull-Doring et al., 2007).

Recent studies suggest that the plant Hsfs also participate in

protective responses to a variety of environmental stresses

(Baniwal et al., 2004; Yokotani et al., 2008). Tomato (Solanum

lycopersicum) plants with suppressed expression of HsfA1a are

extremely sensitive to high temperature stress (Mishra et al.,

2002). A rice spotted leaf gene (Spl7) encodes HsfA4d, and

plants with mutations in this gene develop spontaneous necrotic

lesions due to hypersensitivity to heat and radiation (Yamanouchi

et al., 2002). Arabidopsis plants expressing a dominant-negative

mutant form of HsfA4a are defective in their responses to

oxidative stress (Davletova et al., 2005; Miller and Mittler, 2006).

In this study, we screened for wheat (Triticum aestivum) genes

that confer Cd tolerance to a Cd-sensitive yeast strain and

identified aheat shock transcription factor of classA4 (TaHsfA4a)

as a Cd tolerance factor. We showed that Ta HsfA4a confers

strong Cd tolerance in yeast and rice. Moreover, we also showed

that the knockdown of Os HsfA4a, a rice homolog of Ta HsfA4a,

causes Cd hypersensitivity. Based on these results, we propose

that multiple members of the class A4 heat shock transcription

factor family are involved in cellular responses to Cd in plants.

RESULTS

Ta HsfA4a Plays a Role as Cd Tolerance Gene of Wheat

To identify wheat genes that confer Cd tolerance, we transformed

the YCF1-null yeast strain DTY167 (Dycf1) with a wheat root cDNA

library. Cd sequestration into the vacuole is defective in Dycf1;

thus, this strain does not grow on half-strength SG agar plates

containing 60 to 80 mM CdCl2. We selected colonies that grew in

the presence of 60 to 80 mM CdCl2 and then isolated and

sequenced the respective plasmids. This allowed us to identify

three Cd tolerance genes. Two of them were known genes,

Phytochelatin syntase 1 (PCS1) (Clemens et al., 1999) and Trans-

membrane 20 (TM20) (Kim et al., 2008). The third encoded a 432–

amino acid polypeptidewith homology to heat shock transcription

factors. We named the encoded protein Ta HsfA4a (GenBank

accession number FJ790791), according to the nomenclature

proposedbyNoveretal. (2001).TaHsfA4acontainsaputativeHSF

DNA binding domain (DBD), an oligomerization domain with a

hydrophobic heptad repeat region (HR-A/B), a putative nuclear

localization signal (NLS), a transcriptional activation motif charac-

terized by aromatic, hydrophobic, and acidic amino acid residues

(AHA), and a putative nuclear export signal (NES) (Kotak et al.,

2004; Baniwal et al., 2007) (see Supplemental Figure 1 online).

Dycf1 cells expressing Ta HsfA4a exhibited dramatically en-

hanced growth when compared with Dycf1 cells transformed

with the empty vector (EV) on half-strength SG agar medium

supplemented with 40 mMCdCl2 (Figure 1). The growth of Dycf1

cells expressing Ta HsfA4a were even better than that of wild-

type yeast cells transformed with EV (Figure 1). The heavy metal

tolerance induced by Ta HsfA4a appeared to be selective for Cd.

Ta HsfA4a expression did not significantly enhance yeast toler-

ance to the other heavy metals, namely, lead, zinc, cobalt,

manganese, silver, mercury, or iron (see Supplemental Figure 2

online). The cellular localization of Ta HsfA4awas tested using Ta

HsfA4a fused to green fluorescent protein (GFP-Ta HsfA4a),

which was found in both the nucleus and cytosol in yeast (see

Supplemental Figure 3A online). This is also the case for some

other heat shock transcription factors (Kotak et al., 2004). GFP-

Ta HsfA4a was also found in the nuclei and cytosols of Com-

melina communis guard cells that transiently expressed the

construct after biolistic bombardment (see Supplemental Figure

3B online).

Os HsfA4a Is a Rice Ortholog of Ta HsfA4a

We searched the plant EST database for homologs of TaHsfA4a.

As shown in the phylogenetic tree presented in Figure 2A, the

amino acid sequence of TaHsfA4a is highly similar to those of the

class A4 Hsfs in barley (Hordeum vulgare), sorghum (Sorghum

bicolor), maize (Zea mays), and rice (blue shading in Figure 2A).

However, the class A4 Hsfs of dicot plants such as Arabidopsis

exhibit low levels of similarity to Ta HsfA4a. In rice, Os HsfA4a

(AK109856) and Os HsfA4d (AK100412) show the highest levels

of amino acid sequence similarity to Ta HsfA4a, with 85.7 and

70.1% similarity, respectively. Os HsfA4d is reported as a rice

spotted leaf gene, required for the protection of plants from heat

and light radiation stresses (Yamanouchi et al., 2002).

We investigated whether Ta HsfA4a homologs from rice and

Arabidopsis confer Cd tolerance in the Dycf1 yeast strain. Os

HsfA4a, the closest rice homolog, conferred tolerance to Cd to a

level similar to that of Ta HsfA4a (Figure 2B), suggesting that Os

HsfA4a is an ortholog of Ta HsfA4a in rice. By contrast, Os

HsfA4d–transformed yeast did not exhibit an increase of Cd

tolerance when compared with the EV-transformed control (Fig-

ure 2B). The transformation of yeast cells with the three Arabi-

dopsis genes with the highest homology to Ta HsfA4a, At HsfA4a

(At4g18880), At HsfA4c (At5g45710), and At HsfA5 (At4g13980),

did not confer Cd tolerance (see Supplemental Figure 4 online).

2 of 13 The Plant Cell

The DNA Binding Domain of Ta HsfA4a Is Critical for

Cd Tolerance

To understand the mechanisms via which Ta HsfA4a confers Cd

tolerance, we performed domain swapping between Ta HsfA4a

and Os HsfA4d (a close Ta HsfA4a homolog that does not confer

Cd tolerance in yeast).Wegenerated several chimeric proteins of

Ta HsfA4a and Os HsfA4d and tested their promotion of Cd

tolerance in yeast (Figure 3). TheN-terminal part containing DBD,

the middle part containing HR-A/B and NLS, or the C-terminal

part containing AHA and NES domains of Os HsfA4d were

replaced with the corresponding parts of Ta HsfA4a (Swap I, II,

and III, respectively; Figure 3A). Interestingly, we found that only

Swap I substantially enhanced Cd tolerance in Dycf1 (Figure 3B).

These results suggest that the DBD of Ta HsfA4a is critical for Cd

resistance.

We then compared the amino acid sequences of the DBDs of

Ta HsfA4a and Os HsfA4a with those of other Hsfs that do not

confer Cd tolerance (Figure 4A). Based on the predicted three-

dimensional structures of DBDs (Figure 4B), the region for the

direct binding to DNA is conserved. However, a few amino acids

outside the direct DNA binding regionwere different in TaHsfA4a

and Os HsfA4a when compared with the other Hsfs (Figures 4A

and 4B). The Ser and Trp amino acid residues, which are

Figure 1. Enhanced Cd Tolerance by HsfA4a in Yeast.

(A) Expression of Ta HsfA4a–conferred Cd tolerance in the Dycf1 yeast

mutant. Enhanced growth of Ta HsfA4a–transformed yeast on half-

strength SG-ura agar plates supplemented with 40 mM CdCl2. OD600,

optical density of the yeast suspension at 600 nm.

(B) Time-dependent growth of yeast strains in SG-ura liquid medium

supplemented with 40 mM CdCl2. The data are means and SE from three

independent experiments.

Figure 2. Plant Proteins with Homology to Ta HsfA4a.

(A) The phylogenetic tree was constructed from an alignment of the

amino acid sequences of the N-terminal parts (DBDs and HR-A/B

regions) of Ta HsfA4a and members of the HsfA4 subfamily (Baniwal

et al., 2007). Phylogenetic analyses were conducted using MEGA 4.0

version. The midpoint-rooted phylogenetic tree was constructed by the

neighbor-joining method. We used 21 HsfA4 protein sequences (see

Supplemental Data Set 1 online). The bootstrap values (percentage) of

1000 replicates are shown at the branching points. Ta HsfA4a from

wheat showed a high level of sequence similarity with Hsfs from other

monocots. The accession numbers of the analyzed sequences are

provided at the end of Methods.

(B) Os HsfA4a transformation conferred Cd tolerance to the Dycf1 yeast

mutant, at a level similar to that obtained with Ta HsfA4a. By contrast, Os

HsfA4d transformation did not have this effect. OD600, optical density at

600 nm of the yeast suspension that was spotted onto the plates.

[See online article for color version of this figure.]

Ta HsfA4a Confers Cadmium Tolerance 3 of 13

conserved in the Hsfs without Cd tolerance, were changed to

Ala-31 and Leu-42 in Ta HsfA4a and to Gly-31 and Ala-42 in Os

HsfA4a, respectively (asterisks in Figure 4A). These residues

seemed to be exposed at the opposite surface of the direct DBD

(Figure 4B). To test whether these two amino acids are critical for

Cd tolerance, we substituted Ser-39 and Trp-50 of Os HsfA4d

with Ala (S39A) and Leu (W50L) and then tested whether these

point mutations conferred Cd resistance to Os HsfA4d (Figures

4C and 4D). The Os HsfA4d (W50L) mutant enhanced the growth

of Dycf1 more effectively than Os HsfA4d, while Os HsfA4d

(S39A) did not promote the growth significantly. However, the

combination of the W50L and S39A mutations conferred a level

of Cd tolerance that was comparable with that of Ta HsfA4a

(Figures 4C and 4D). These results indicate that Ala-31 and Leu-

42 in the DBD domain of Ta HsfA4a are critical for its ability to

confer Cd tolerance.

CUP1 Is Necessary for Ta HsfA4a–Mediated Cd Resistance

As a transcription factor, HsfA4a is expected to function by

regulating the expression levels of other genes. To identify the

target genes of HsfA4a, we investigated whether any of themany

genes known to be involved in stress responses in yeast (Vido

et al., 2001) were upregulated in yeast cells overexpressing Ta

HsfA4a. The genes tested are listed in Supplemental Table

1 online. Among them,CTT1 (Cytosolic catalase T),CUP1,GRE1

(Genes de Respuesta a Estres), HSP12 (Heat Shock Protein 12),

HSP26, and SSA4 (Stress-Seventy subfamily A4) were most

highly upregulated in Ta HsfA4a–expressing yeast (see Supple-

mental Table 1 online). The individual expression of these six

genes in yeast cells revealed that only CUP1 (which encodes

metallothionein) improved Cd tolerance substantially.CUP1was

previously reported to confer Cu and Cd tolerance (Jeyaprakash

et al., 1991); thus, we further tested whether CUP1 is a target of

HsfA4a (Figure 5). First, we examined if Ta HsfA4a induces CUP1

(Figure 5A). Ta HsfA4a overexpression increased the levels of

expression of the CUP1 transcript in wild-type yeast cells (which

carry two copies of theCUP1 locus) by about fourfold (Figure 5A;

see Supplemental Figure 5 online). By contrast, expression of the

rice homolog Os HsfA4d, which does not increase Cd tolerance,

could not induce CUP1 expression (see Supplemental Figure 5

online). We then tested whether CUP1 is required for the Ta

HsfA4a–mediated Cd tolerance (Figure 5B). The Dcup1 mutant,

which does not carry any copy of CUP1, is highly sensitive to Cd

(Figures 5A and 5B) and does not grow in the presence of CdCl2at concentrations as low as 3 mM. Ta HsfA4a overexpression in

this mutant did not enhance Cd tolerance of the yeast cells,

which remained sensitive to 3mMCdCl2 (Figure 5B). By contrast,

Figure 3. Characterization of Ta HsfA4a via Domain Swapping with Os HsfA4d.

(A) Schematic representation of the stratagy used for domain swapping between Ta HsfA4a and Os HsfA4d. Chimeric proteins were generated by

replacing the N-terminal part containing DBD, the middle part containing HR-A/B and NLS, or the-C-terminal part containing AHA and NES domains of

Os HsfA4d with the corresponding parts of Ta HsfA4a. SwapI, DBD; SwapII, HR-A/B and NLS; SwapIII, AHA and NES domain.

(B) Cd tolerance test in Dycf1 yeast cells transformed with domain-swapped mutants on half-strength SG-ura agar plates supplemented with 40 mM

CdCl2. SwapI alone exhibited Cd resistance comparable with that of Ta HsfA4a. SwapII and SwapIII did not improve Cd tolerance significantly.


expression of Ta HsfA4a in theCUP1 single-copy mutant (cup1s)

caused an increase in the level of CUP1 expression and enabled

the cup1s strain to grow on 3 mM CdCl2-containing medium

(Figure 5B). These results suggest that CUP1 contributes to the

Ta HsfA4a–induced Cd tolerance by acting as a downstream

target of HsfA4a.

Overexpression of Ta HsfA4a Enhances Cd Tolerance

in Rice

To investigate whether HsfA4a confers Cd tolerance in planta, Ta

HsfA4a was expressed in rice under the control of the maize

ubiquitin promoter. Because of technical difficulties in generating

transgenic wheat, we chose to use rice, which is one of the most

important crops and is a well-established model plant. The

growth of wild-type rice and two independent rice lines over-

expressing Ta HsfA4a (Ta HsfA4a OX1 and OX2) was compared

on half-strength Murashige and Skoog (MS) agar supplemented

with 0, 100, 200, or 300 mM CdCl2 for 2 weeks (Figures 6A and

6B). The Ta HsfA4a OX rice plants were slightly shorter than

isogenic wild-type plants when grown in control conditions

(Figure 6A, left panels). By contrast, the Ta HsfA4a OX rice plants

were significantly taller than isogenic wild-type specimens when

cultured inmedia containing Cd (100, 200, or 300mM) (Figure 6A,

Figure 4. Identification of Amino Acid Residues in the DBD of Ta HsfA4a That Are Critical for Cd Tolerance.

(A) Amino acid sequence alignment of the DBD of Ta HsfA4a with those of the DBDs of its rice homologs. The two variant amino acids that are not found

in distantly related Hsfs and that may be important for Ta HsfA4a–mediated Cd tolerance are marked with an asterisk. Black, dark-gray, gray, and white

shadings indicate identical, conservative, weakly similar, and nonsimilar amio acids, respectively.

(B) Predicted three-dimensional structures of the DBDs of Ta HsfA4a and Os HsfA4d. The blue-colored region represents the site of direct DNA

interaction. The two critical amino acids are indicated in pink (Ta HsfA4a) or green (Os HsfA4d). Note that these residues seem to be exposed at the

surface opposite to the direct DNA binding region.

(C) Substitution of the two amino acids bestowing Cd tolerance to Os HsfA4d. The S39A and W50L in combination conferred Cd resistance to Os

HsfA4d comparable to Ta HsfA4a.

(D) Time-dependent growth of yeast strains in SG-ura liquid medium supplemented with 40 mM CdCl2. The double mutation (S39A and W50L)

significantly enhanced the growth of Dycf1, compared with either of the S39A or W50L single mutations. The data are means and SE from three

independent experiments.


right panels; see quantification in Figure 6B). These results

demonstrate that Ta HsfA4a confers Cd tolerance in rice. We

also testedwhether TaHsfA4a expression conferred tolerance to

other heavy metals, such as Pb and Zn. The Ta HsfA4a OX1 rice

plants did not differ in size from wild-type plants when grown in

media containing 2.5 mM Pb(NO3)2 or 5 mM ZnCl2. Overall plant

growth was compromised in these media (see Supplemental

Figure 6 online).

Next, we wondered whether HsfA4a also upregulates metal-

lothionein in rice, as it did in yeast. Among the 11 metallothio-

neins described in rice, we speculated thatMT-I-1a is a potential

target gene of HsfA4a because the promoter region of MT-I-1a

has a heat shock element (Zhou et al., 2006). We measured the

expression levels of the MT-I-1a transcript in rice plants trans-

formed with Ta HsfA4a using quantitative real-time PCR. Ta

HsfA4a–transformed plants exhibited higher expression levels of

the MT-I-1a transcript than wild-type plants (Figure 6C). This

result suggests that HsfA4a confers Cd resistance in rice via

upregulation of metallothionein, at least in part.

The Knockdown of Os HsfA4a in Rice Results in

Cd Sensitivity

If Ta HsfA4a and its orthologs confer Cd tolerance in plants,

their loss-of-function mutations might cause increases in Cd

sensitivity. Thus, we investigated whether rice plants with loss-

of-function HsfA4a mutations are more sensitive to Cd than

wild-type plants. We obtained two T-DNA insertional mutant

rice lines with insertions in the Os HsfA4a gene (3A-13297 and

2C-70146; the rice-flanking sequence tag database; http:://

www.postech.ac.kr/life/pfg) and tested their growth inmedium

containing 150 mMCdCl2 (Figure 7; see Supplemental Figure 7

online). The first allele (hsfA4a-1, 3A-13297) had a T-DNA

insertion in the promoter region (2189 bp from the start codon;

Figure 7A), which reduced the Os HsfA4a transcript level to

;20% of that of the wild type (Figure 7B). The growth of

hsfA4a-1 rice plants was similar to that of wild-type plants in

control medium but was more severely reduced than that of

wild-type plants in medium containing CdCl2 (Figure 7C). The

mean height and fresh weight of hsfA4a-1 plants were 71.9%

(Figure 7D) and 72.6% (Figure 7E), respectively, compared

with those of the wild type in the same CdCl2-containing

medium.

The second allele (hsfA4a-2, 2C-70146), which had a T-DNA

insertion at the end of the second exon, produced no full-length

transcripts but partial ones with a premature stop codon (see

Supplemental Figures 7A and 7B online). In contrast with the first

allele, the growth of the second mutant did not differ from that of

wild-type plants in either control medium or medium containing

150 mM CdCl2 (see Supplemental Figure 7C online), suggesting

that the truncated Os HsfA4a in the hsfA4a-2 rice plants was

functional. Consistent with this explanation, the C-terminal trun-

cated mutant protein Os HsfA4a (D106 amino acids) conferred

Cd tolerance toDycf1 yeast, to a level only slightly lower than that

conferred by the intact Os HsfA4a (see Supplemental Figure 8

online). These results further support our conclusions from the

domain-swapping experiment where it was found that the DBD,

not the C-terminal region, is critical for Cd tolerance (Figure 3).

Figure 5. CUP1-Mediated Cd Resistance of Ta HsfA4a in Yeast.

(A) RNA gel blot analysis of CUP1 expression. Ta HsfA4a upregulated CUP1 in wild-type yeast cells and in the CUP1 single-copy mutant (cup1s). rRNA

was presented as loading control.

(B) The Ta HsfA4a–induced Cd tolerance was dependent on CUP1 expression. Ta HsfA4a transformation conferred Cd tolerance in the cup1s yeast

mutant but not in the cup1 deletion mutant (Dcup1) on half-strength SG-ura agar plates supplemented with 3 mM CdCl2.


Taken together, our results demonstrate that HsfA4a indeed

plays an important role in Cd tolerance in rice.

Cd Induces MT Expression in Rice andWheat

To further test the in vivo function of HsfA4a, we examined

whether Cd stress induces the expression of Ta HsfA4a, its rice

homolog, and their target genes (MTs) in rice and wheat. As

putative targets of HsfA4a in wheat, we chose Ta MT-I-1

(CD373636) and Ta MT-I-2 (CD454237), which are the closest

homologs of Os MT-I-1a. Rice and wheat seedlings grown on

half-strength MS agar medium for 7 d were transferred to half-

strength MS liquid medium. After 12 h of incubation, the seed-

lings were exposed to 100 mM CdCl2 for 2 h, and the transcript

levels of target genes were analyzed using quantitative real-time

PCR (Figure 8). Cd treatment increasedOsHsfA4a expression by

more than fourfold in roots of rice plants, compared with ex-

pression in the nontreated controls (Figure 8A). Cd treatment also

increased the expression of Os MT-I-1a in rice roots, to a level

comparable with that induced by Ta HsfA4a overexpression (cf.

Figures 8A and 6C). Cd treatment did not increase Os HsfA4a or

OsMT-I-1a expression in shoots, suggesting that the site where

HsfA4a confers Cd tolerance is the root and not the shoot. In

wheat, we observed a similar pattern of induction of HsfA4a

and its putative target genes; Cd treatment caused dramatic

Figure 6. Enhanced Cd Resistance in Ta HsfA4a–Overexpressing Rice Plants.

Ta HsfA4a was overexpressed in rice plants (O. sativa Dongjin). The growth of two independent Ta HsfA4a–overexpressing rice lines (Ta HsfA4a OX1

and OX2) was tested over a period of 2 weeks on half-strength MS agar plates supplemented with 0, 100, 200, or 300 mM CdCl2. We used segregated

wild-type rice as a control.

(A) Representative images of 2-week-old wild-type, Ta HsfA4a OX1, and Ta HsfA4a OX2 lines in the presence and absence of 200 mM CdCl2. Bars = 3

cm.

(B) Quantitative measurement of the height of Ta HsfA4a–overexpressing plants (mean6 SE, n = 36). Ta HsfA4a OX rice seedlings were taller than wild-

type control plants in the presence of Cd (Student’s t test, *P < 0.05 and **P < 0.01). Results are representative of three independent experiments.

(C) Upregulation of Os MT-I-1a in Ta HsfA4a OX rice plants. We measured the levels of expression of the Os MT-I-1a transcript in Ta HsfA4a–

transformed plants using quantitative real-time PCR. Two independent Ta HsfA4a–transformed lines (TaHsfA4a OX1 and OX2) expressed OsMT-I-1a at

a level that was higher than that of the wild-type control. Data represent two independent experiments (mean 6 SD).


increases in the transcript levels of TaHsfA4a, TaMT-I-1, and Ta

MT-I-2 in the roots (Figure 8B). These results show thatHsfA4a is

upregulated by Cd treatment and that it in turn upregulates the

expression of target genes, including MTs, and thereby confers

Cd tolerance in planta.

DISCUSSION

In this study, we identified and characterized two orthologs of a

heat shock transcription factor that is implicated in Cd tolerance

in wheat and rice. We demonstrate that a wheat Hsf A4a

Figure 7. The Cd-Sensitive Phenotype of hsfA4a-1 Knockdown Rice Plants.

(A) The site of T-DNA insertion in the hsfA4a-1 plants.

(B) The expression level of Os HsfA4a in the hsfA4a-1 knockdown plants. Os HsfA4a transcript levels were tested using RT-PCR and measured using

quantitative real-time PCR. The hsfA4a-1 knockdown rice expressed Os HsfA4a transcript at around 20% of the wild-type levels. Data represent three

independent experiments (mean 6 SE).

(C) Photographs of representative wild-type and hsfA4a-1 knockdown plants in the presence and absence of 150 mM CdCl2. Bars = 2 cm.

(D) and (E) Quantitative measurements of plant height (D) and fresh weight (E) of wild-type and hsfA4a-1 knockdown plants. The hsfA4a-1 plants were

smaller than the wild-type control plants (Student’s t test, *P < 0.01). Results represent three independent experiments (mean 6 SE, n = 36).


(Ta HsfA4a) confers strong Cd tolerance in yeast and rice and

that this tolerance is mediated at least in part by metallothionein.

The rice ortholog Os HsfA4a confers similar Cd tolerance to

yeast, while its loss-of-function mutation causes Cd hypersen-

sitivity in rice plants. We propose that HsfA4a functions in Cd

tolerance in planta becauseHsfA4a genes are upregulated byCd

stress, and the HsfA4a proteins in turn activate transcription of

MT genes, resulting in improved Cd tolerance in wheat and rice.

We have shown that the Ta HsfA4a–inducedMT expression is

crucial for Ta HsfA4a–mediated Cd tolerance in yeast. Metallo-

thionein is a well-known chelator of Cd/Cu both in yeast and in

rice (Ecker et al., 1986; Jin et al., 2006). Expression of Ta HsfA4a

inducedCUP1 expression in yeast and OsMT-I-1a expression in

rice (Figures 5A and 6C), while deletion of the metallothionein

gene abolished the Ta HsfA4a–mediated Cd tolerance in yeast

(Figure 5B). Moreover, in response to Cd stress, OsMT-I-1a and

wheat MT genes were transcriptionally induced in the roots,

where the expressions of Ta HsfA4a and Os HsfA4a were also

highly induced (Figure 8). These results indicate that the MT

genes are important targets of HsfA4a and that MT expression

may be necessary for Cd tolerance in plants.

Althoughmetallothionein was found to be important for the Cd

tolerance functions of Ta HsfA4a, it is not likely to be the sole

target of HsfA4a-regulated Cd tolerance, as the overexpression

of CUP1 alone was not sufficient to induce the full range of Cd

tolerance conferred by Ta HsfA4a (D. Shim and Y. Lee, unpub-

lished data); therefore, HsfA4a most likely activates multiple Cd

tolerance mechanisms, similarly to the mechanisms described

for MTF-1 in animal cells (Egli et al., 2003; Wimmer et al., 2005).

The HsfA4a-induced Cd tolerancemechanismsmay encompass

several major pathways (each of which may exert significant

effects) or may assemble many targets that contribute minor

effects. To address this question, it will be necessary to analyze

the differential gene expression patterns present in HsfA4a-

expressing and wild-type yeast and rice cells at the whole-

genome level. This approach will also enable the identification of

novel targets of HsfA4a, which may uncover new aspects of Cd

tolerance.

Previously, several transcription factors have been reported to

be induced by heavy metal stress in plants; however, their

physiological roles in heavy metal tolerance have not been

clearly demonstrated (Herbette et al., 2006). A few transcriptional

factors, such as an ERF/AP2 family member, were reported to

enhance heavy metal resistance when overexpressed (Tang

et al., 2005); however, the tolerance was mediated by protection

from oxidative damage, which is implicated in general stress

tolerance. HsfA4a is unique not only in the high level of tolerance

that it confers, but also in its Cd specificity. Ta HsfA4a greatly

enhanced Cd tolerance in yeast and rice, but did not significantly

enhance their tolerance to the other heavy metals, namely, lead,

zinc, cobalt, manganese, silver, mercury, and iron (see Supple-

mental Figures 2 and 6 online). Nor did Ta HsfA4a confer any

tolerance to heat stress in yeast or rice, although its gene

expression was induced by heat stress to levels comparable

with those induced by Cd stress (D. Shim and Y. Lee, unpub-

lished data). An intriguing question remains regarding the mech-

anisms underlying the Cd specificity of the HsfA4a-induced

response. HsfA4a might activate target genes that are specifi-

cally involved in Cd tolerance. Metallothionein is such a potential

target gene because it is a good chelator of Cd and Cu but is not

effective in the detoxicification of other heavymetals (Ecker et al.,

1986). Supporting this explanation, transcripts of CUP1 were

elevated in yeast cells transformed with Ta HsfA4a but not with

Os HsfA4d (see Supplemental Figure 5 online). Future efforts to

identify other genes regulated by HsfA4a will provide further

insight on this matter.

Among the class A4 Hsfs we tested, Ta HsfA4a andOs HsfA4a

conferred Cd tolerance to yeast and plants, but other Hsfs with

Figure 8. Induction ofHsfA4a andMTGenes in Roots of Wheat and Rice

in Response to Cd.

(A) Transcript levels of Os HsfA4a and Os MT-I-1a were increased in the

roots of rice seedlings after Cd treatment.

(B) Transcript levels of Ta HsfA4a, Ta MT-I-1, and Ta MT-I-2 were

increased in the roots of wheat seedlings after Cd treatment. Seven-day-

old wheat and rice seedlings grown on half-strength MS agar medium

were transferred to half-strength MS liquid medium for 12 h (nontreated

control, Con) and then exposed to 100 mM CdCl2 for 2 h (Cd). All

transcript levels were assessed by quantitative real-time PCR. The

relative expression values to the control were calculated as DDCT.

G3PDH and Act1 were analyzed as a constitutively expressed control

gene in wheat and rice, respectively. Data represent three independent

experiments (mean 6 SE, n = 9).


similar basic structures (Os HsfA4d, At HsfA4a, and At HsfA4c)

did not (Figure 2B; see Supplemental Figure 4 online). Our

detailed analyses of the functional domains of these proteins

revealed that the DBD is critical for Cd tolerance and that the

alteration of only two amino acids in the DBD region could

transform an Hsf ineffective in Cd tolerance into an effective one

(Figures 3 and 4). The question of how these slight differences in

the DBD determine the physiological functions of these Hsfs is a

very intriguing issue. The three-dimensional structure of the DBD

predicted that the two critical amino acids are exposed at the

opposite surface of the DNA binding site (Figure 4B), which

indicates they are not directly involved in the interaction with cis-

acting elements. Therefore, we speculate that they might be

involved in the interaction with other factors, such as activators

or enhancers, and thereby promote the expression of Cd toler-

ance genes. The identification of the interacting partner(s) of

HsfA4a, in particular of those that interact with the two critical

amino acids, may reveal interesting new features of the Cd

tolerance mechanism. Alternatively, those two amino acids may

somehow induce a conformational change in the DBD and

consequently alter the binding affinity of the protein for cis-

acting elements in the CUP1 promoter. Extensive studies of the

cis-acting elements that bind to HsfA4a will be needed for us to

understand the mode of action of the transcription factor in

activating target genes.

It is interesting to note that, instead of an MTF-1-like tran-

scription factor in animal cells, we found HsfA4a orthologs as Cd

tolerance genes in wheat and rice plants. Putative metal-respon-

sive elements have been described in the promoter regions of

several plant genes, which include the bean metal-responsive

gene SR2 and the rice metallothionein genes (Zhou et al., 2006;

Qi et al., 2007). However, there are no reports of plant genes with

a high amino acid sequence homology toMTF-1. In plant, unique

metal-responsive transcription factor(s) that are structurally dis-

tinct from, but functionally similar to, animal MTF-1 may function

in the control of heavy metal homeostasis. Our identification of

HsfA4a as a Cd tolerance factor suggests that Hsfs may be

potential substitutes for MTF-1 in plants. Plant genomes contain

many Hsf genes, and there is limited information available

regarding the physiological roles of plant Hsfs. For example,

tomato HsfA1a and Arabidopsis HsfA2 are required for inducing

thermotolerance (Mishra et al., 2002; Ogawa et al., 2007), rice

HsfA4d is reported to be an anti-apoptotic factor (Yamanouchi

et al., 2002), Arabidopsis HsfA4a participates in responses to

oxidative stress (Davletova et al., 2005; Miller and Mittler, 2006),

and Arabidopsis HsfA9 has a role in seed development (Kotak

et al., 2007). We now add Ta HsfA4a and Os HsfA4a as Cd

tolerance factors to this limited list of functionally characterized

Hsfs. There remains the possibility that Hsfs from different

subfamilies, and/or different types of transcription factors, also

participate in the Cd stress responses, since recent studies of

transcript profiles in plants show that diverse transcription fac-

tors, includingHsfs, are upregulated byCd stress (Herbette et al.,

2006; Weber et al., 2006).

The evolutionary importance of plant Hsfs is an important area

of study since, as we mentioned previously, plant genomes

contain far more Hsf genes than yeast or animal genomes. It is

tempting to speculate that plants may have evolved functionally

specialized Hsfs to respond to a variety of stresses. For example,

some class A4 members might have evolved to confer Cd

tolerance via a few amino acid changes in their DBDs (e.g.,

Ala-31 and Leu-42 in Ta HsfA4a; Gly-31 and Ala-42 in Os

HsfA4a). The yeast Hsf (HSF1) provides an example of the

acquisition of a new function via a single amino acid change in

the DBD. Normally, HSF1 does not activate the CUP1 gene;

however, a mutation of Tyr to Phe in the DBD of HSF1 allowed it

to activate the CUP1 gene and thereby confer resistance to Cd

and Cu (Sewell et al., 1995). We further speculate that the novel

function of class A4 Hsfs as Cd tolerance factors evolved after

the evolutionary split between monocot and dicot plants be-

cause we could not find any dicot genes that are highly homol-

ogous to Ta HsfA4a in their DBDs (Figure 2A).

In conclusion, we have demonstrated the function of two

orthologs of the plant class A4Hsfs, which confer Cd tolerance in

planta. The Cd-responsive induction of the Ta HsfA4a and Os

HsfA4a genes was rapid and strong in wheat and rice roots, and

their expression induced a well-known Cd tolerance gene,

metallothionein, leading to Cd tolerance in the plants. Further

studies aimed at identifying and characterizing other target

genes and their promoters will be needed to enhance our

understanding of the modes of action of these Hsfs. It will also

be important to study the upstream signaling pathways that

connect Cd stress to activation of the Hsfs. We expect that our

findings will contribute to bioengineering approaches aimed at

the development of Cd resistant plants. For example, expression

of HsfA4a in graminaceous plants (e.g., reeds) may be beneficial

for the phytoremediation of Cd-contaminated wetlands.

METHODS

Growth Conditions of Rice Plants

Seeds of wild-type rice (Oryza sativaDongjin), Ta HsfA4a–overexpressing

rice lines (Ta HsfA4a OX1 and Ta HsfA4a OX2), and hsfA4a-1/2 were

surface-sterilized and germinated on half-strength MS medium contain-

ing 1.5% sucrose, 0.8% phytoagar, and 0.28 mM myoinositol (Sigma-

Aldrich), in the presence or absence of 100 to 300 mM CdCl2, 100 mM

CuCl2, 3 mM Pb(NO3)2, and 5 mM ZnCl2. Seedlings were grown for 7 d at

278C under continuous light and were then transferred to soil in a

greenhouse and raised to maturity.

Isolation of Cd Tolerance Genes from aWheat Root cDNA Library

For the identification of Cd tolerance genes from a wheat (Triticum

aestivum) root cDNA library, we introduced the library into the ycf1-null

and Cd-sensitive mutant yeast strain DTY167 (MATa ura3-52 his6 leu2-

3,-112 his3-D200 trp1-901 lys2-801 suc2-D, ycf1::hisG) using the lithium

acetate method (Ito et al., 1983). Colonies that grew on medium

containing 60 to 80 mMCdCl2 were selected, and plasmids were isolated

from these cells. The Cd tolerance function of the isolated plasmids was

confirmed by repeating the transformation of the Dycf1 cells.

Isolation of HsfA4a Knockdown Plants

Two Os HsfA4a T-DNA insertional mutants were identified in the rice-

flanking sequence tag database (http:://www.postech.ac.kr/life/pfg). T2

progeny of the primary insertional mutants were grown to maturity to

amplify the seeds. The genotypes of the progeny were determined by


PCR with three primers for each line. For the hsfA4a-1 line (3A-13297),

two Os HsfA4a–specific primers (Os HsfA4a F1and Os HsfA4a R1), and a

T-DNA–specific primer (LB-2) were used. For the hsfA4a-2 line (2C-

70146), the hsfA4a-2–specific primers (Os HsfA4a F2 and Os HsfA4a R2)

and the T-DNA-specific primer (LB-2) were used. The primer sequence

information is provided in Supplemental Table 2 online.

Generation of Mutant Constructs of HsfA4d

All domain-swapping mutants of Os HsfA4d were generated using a

two-step PCR approach. The Ta HsfA4a gene fragments encoding the

N-terminal fragment containing DBD (P1), themiddle fragment containing

HR-A/B and NLS (P2), or the C-terminal fragment containing AHA and

NES (P3) were synthesized by PCR fromplasmid pYES2-Ta HsfA4a using

the following gene-specific primer sets: Ta HsfA4a_F1 and Ta HsfA4a_R1

for P1, Ta HsfA4a-F2 and Ta HsfA4a_R2 for P2, and Ta HsfA4a_F3 and Ta

HsfA4a_R3 for P3. The gene fragments of Os HsfA4d were generated

from pYES2-Os HsfA4d using gene-specific primer sets. The DNA

sequence for the N-terminal fragment containing DBD (P4) was synthe-

sized using OsHsfA4d_F1 andOs HsfA4d_R1. The DNA sequence for the

fragment containing DBD, HR-A/B, and NLS (P5) was synthesized using

Os HsfA4d_F1 and Os HsfA4d-R2. To generate DNA sequence for the

fragment containing HR-A/B, NLS, AHA, and NES (P6), we used the PCR

primers Os HsfA4d_F2 andOs HsfA4d_R3. To generate DNA sequence for

the C-terminal fragment containing AHA and NES (P7), we used the PCR

primers Os HsfA4d_F3 and Os HsfA4d_R3. The entire DNA sequence of

Swap I was generated by a secondPCR from themix of P1 and P6 using Ta

HsfA4a_F1 andOsHsfA4d_R3. TheSwap III DNAwas synthesized from the

mix of P3 and P5 using Os HsfA4d_F1 and Ta HsfA4a_R3. For the

generation of the Swap II mutant, the chimeric DNA sequence of P2 and

P7 was first synthesized and then the entire DNA sequence was generated

using Os HsfA4d_F1, Ta HsfA4a_F2, and Os HsfA4d_R3. To assess Cd

tolerance in yeast cells, we subcloned the full-length DNA sequences of the

Swap mutants into the BamHI and XhoI sites of the pYES2 vector. Finally,

weusedsite-directedmutagenesis to generate the aminoacid substitutions

in theDBDofOsHsfA4d (S39A andW50L) (Braman et al., 1996). The fidelity

of the DNA sequence of all constructs was verified by sequencing. The

primer sequence information is provided in Supplemental Table 2 online.

Analysis of Cd Tolerance in Yeast

The Dycf1, cup1s (MATa, trp1-1, leu2-3, leu2-112, gal1, ura3-50, His-,

cup1 single copy), and Dcup1 (MATa, trp1-1, leu2-3, leu2-112, gal1, His-,

ura3-50, cup1delta::ura3) yeast strains and their isogenic wild-type

counterparts were transformed with the indicated constructs using the

lithium acetatemethod. Transformantswere selected onminimalmedium

lacking uracil. To perform theCd tolerance test, yeast lines expressing the

indicated constructs were grown on half-strength SG agar plates in the

absence or presence of 3 to 40 mM CdCl2 at 308C for 3 d.

Gene Expression Analysis

Total RNA was extracted from wheat and rice seedlings and from yeast

cells using the TRIzol reagent. For RNA gel blot analysis of CUP1

expression, yeast cells were grown on half-strength synthetic galac-

tose-uracil (SG-ura) liquid medium for 3 d. Subsequent RNA preparation

and RNA gel blot hybridization were performed as described previously

(Sambrook and Russell, 2001). For quantitative expression analysis of

Hsfs andMTs in wheat and rice plants, total RNA (3 mg) was subjected to

cDNA synthesis using a Powerscript RT kit (Clontech) according to the

manufacturer’s instructions. Quantitative real-timePCRwasperformed in

a Thermal Cycler Dice real-time system (Takara) using the SYBR Premix

Ex Taq kit (Takara) according to the manufacturer’s instructions. The Ta

HsfA4a cDNA was amplified using the following specific primer pair: Ta

HsfA4a_RT_F and Ta HsfA4a_RT_R. Glyceraldehyde-3-phosphate de-

hydrogenase (G3PDH) was used as an internal control in PCR amplifica-

tion, and its cDNA was amplified using the following gene-specific

primers: Ta G3PDH_F and Ta G3PDH_R. In the case of rice, the Os

HsfA4a cDNA was amplified using the following specific primer pair: Os

HsfA4a_RT_F and Os HsfA4a_RT_R. The rice Actin1 (Os Act1) gene was

used as an internal control with the primers Os Act1_F and Os Act1_R.

The relative expression levels of the rice MT-I-1a gene (Os MT-I-1a) and

the wheat homologs (Ta MT-I-1 and Ta MT-I-2) were analyzed by

quantitative real-time PCR using the following specific primer pairs: Os

MT-I-1a_RT_F andOsMT-I-1a_RT_R, TaMT-I-1_F and TaMT-I-1_R, and

Ta MT-I-2_F and Ta MT-I-2_R (for the primer sequence information, see

Supplemental Table 2 online). Transcript levels were calculated relative to

the controls and were expressed as DDCT. Data represent means and

standard errors of three replicates. To find the candidate target genes of

Ta HsfA4a, the expression levels of the yeast stress-responsive genes

were analyzed by quantitative real-time PCR using the gene-specific

primers listed in Supplemental Table 1 online.

Accession Numbers

The names (with accession numbers) of sequences used in the

phylogenetic analysis are as follows: Arabidopsis thaliana (At)

HsfA4a (At4g18880), HsfA4c (At5g45710); Citrus sinensis (Cs) HsfA4a

(DY270414);Glycinemax (Gm) HsfA4a (TC135284);Hordeum vulgare (Hv)

HsfA4a (TC42448); Lycopersicon esculentum (Le) HsfA4a (BT014619)

andHsfA4b (TC107140); Lotus japonicus (Lj) HsfA4a (AP004978); Lactuca

sativa (Ls) HsfA4a (DY973605);Medicago sativa (Ms) HsfA4a (AF494082);

Medicago truncatula (Mt) HsfA4a (TC79769); Nicotiana tabacum (Nt)

HsfA4a (AB014484); Oryza sativa japonica (Os) HsfA4a* (AK109859) and

HsfA4d (AK100412);Phaseolus aureus (Pa) HsfA4a (AY052627);Sorghum

bicolor (Sb) HsfA4a (BM322601); Solanum tuberosum (St) HsfA4b

(BG591987); Triticum aestivum (Ta) HsfA4d (CV766704); Taraxacum

officinarum (To) HsfA4a (DY824833); and Zea mays (Zm) HsfA4a

(NP003871). For references to the ESTs and genomic clones used,

please refer to Baniwal et al. (2007). *Os HsfA4a is also known as

OsHsfA4b in some publications. In this article, we follow the nomencla-

ture suggested by Kotak et al. (2004) and Baniwal et al. (2007).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Ta HsfA4a Has the Conserved Structure of

Hsfs.

Supplemental Figure 2. Expression of Ta HsfA4a Did Not Enhance

Tolerance of Other Heavy Metals in Yeast.

Supplemental Figure 3. GFP-Ta HsfA4a Is Localized to Both the

Nucleus and Cytosol in Yeast and Plant Cells.

Supplemental Figure 4. Arabidopsis Genes Homologous to

Ta HsfA4a Did Not Confer Cd Tolerance to Dycf1 Yeast.

Supplemental Figure 5. CUP1 mRNA Levels Were Increased in

Yeast Cells Expressing Ta HsfA4a but Not in the Same Strain of Yeast

Expressing Os HsfA4d.

Supplemental Figure 6. Ta HsfA4a–Expressing Rice Plants Showed

Similar Growth to Wild-Type Plants in Media Containing Lead or Zinc.

Supplemental Figure 7. Rice Plants with a T-DNA Insertion at the

C-Terminal End of Os HsfA4a (Os HsfA4a-2) Produced Truncated Os

HsfA4a Transcripts and Did Not Differ in Growth from Wild-Type

Plants under Control and Cd Stress Conditions.

Supplemental Figure 8. The C-Terminal Truncated Os HsfA4a (from

hsfA4a-2) Conferred Cd Tolerance to Dycf1 Yeast.


Supplemental Table 1. List of the Yeast Stress-Responsive Genes

Tested in This Study.

Supplemental Table 2. Sequences of Primers Used in This Study.

Supplemental Data Set 1. Text File of the Alignment Used for the

Phylogenetic Analysis Shown in Figure 2A.

ACKNOWLEDGMENTS

We thank Julian Schroeder for providing the wheat root cDNA library,

Dennis Thiele for providing the ycf1 null mutant yeast, and Sun-Hee

Leem for providing the cup1s and cup1 null mutant yeast. The primers

used in real-time PCR were generously provided by Eun-Woon Noh’s

laboratory at the Korea Forest Research Institute. We also thank

Aekyung Han and Jong Soon Kim for maintenance of the rice plants.

This work was supported by grants from the Global Research Program

of the National Research Foundation of Korea (K20607000006-

08A0500-00610 to Y.L. and E.M.), from the Ministry of Education

Science and Technology/National Research Foundation of Korea to

the Environment Biotechnology National Core Research Center (Grant

20090091490), Korea, to Y.L., from the Crop Functional Genomic Center,

the 21st Century Frontier Program (Grant CG1111 to G.A.), and by the

Basic Research Promotion Fund from Korea Research Foundation (KRF-

2007-341-C00028 to G.A.).

Received March 12, 2009; revised November 16, 2009; accepted No-

vember 30, 2009; published December 22, 2009.

REFERENCES

Alscher, R.G., Erturk, N., and Heath, L.S. (2002). Role of superoxide

dismutases (SODs) in controlling oxidative stress in plants. J. Exp.

Bot. 53: 1331–1341.

Baniwal, S.K., et al. (2004). Heat stress response in plants: a complex

game with chaperones and more than twenty heat stress transcription

factors. J. Biosci. 29: 471–487.

Baniwal, S.K., Chan, K.Y., Scharf, K.D., and Nover, L. (2007). Role of

heat stress transcription factor HsfA5 as specific repressor of HsfA4.

J. Biol. Chem. 282: 3605–3613.

Braman, J., Papworth, C., and Greener, A. (1996). Site-directed

mutagenesis using double-stranded plasmid DNA templates.

Methods Mol. Biol. 57: 31–44.

Butt, T.R., and Ecker, D.J. (1987). Yeast metallothionein and applica-

tions in biotechnology. Microbiol. Rev. 51: 351–364.

Chen, T., and Parker, C.S. (2002). Dynamic association of transcrip-

tional activation domains and regulatory regions in Saccharomyces

cerevisiae heat shock factor. Proc. Natl. Acad. Sci. USA 99: 1200–

1205.

Clemens, S. (2001). Molecular mechanisms of plant metal tolerance and

homeostasis. Planta 212: 475–486.

Clemens, S., Kim, E.J., Neumann, D., and Schroeder, J.I. (1999).

Tolerance to toxic metals by a gene family of phytochelatin synthases

from plants and yeast. EMBO J. 18: 3325–3333.

Clemens, S., Palmgren, M.G., and Kramer, U. (2002). A long way

ahead: Understanding and engineering plant metal accumulation.

Trends Plant Sci. 7: 309–315.

Dalton, T.P., Solis, W.A., Nebert, D.W., and Carvan III, M.J. (2000).

Characterization of the MTF-1 transcription factor from zebrafish

and trout cells. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 126:

325–335.

Davletova, S., Rizhsky, L., Liang, H., Shengqiang, Z., Oliver, D.J.,

Coutu, J., Shulaev, V., Schlauch, K., and Mittler, R. (2005). Cyto-

solic ascorbate peroxidase 1 is a central component of the reactive

oxygen gene network of Arabidopsis. Plant Cell 17: 268–281.

Ecker, D.J., Butt, T.R., Sternberg, E.J., Neeper, M.P., Debouck, C.,

Gorman, J.A., and Crooke, S.T. (1986). Yeast metallothionein func-

tion in metal ion detoxification. J. Biol. Chem. 261: 16895–16900.

Egli, D., Selvaraj, A., Yepiskoposyan, H., Zhang, B., Hafen, E.,

Georgiev, O., and Schaffner, W. (2003). Knockout of ’metal-respon-

sive transcription factor’ MTF-1 in Drosophila by homologous recom-

bination reveals its central role in heavy metal homeostasis. EMBO J.

22: 100–108.

Goyer, R.A. (1997). Toxic and essential metal interactions. Annu. Rev.

Nutr. 17: 37–50.

Ha, S.B., Smith, A.P., Howden, R., Dietrich, W.M., Bugg, S.,

O’Connell, M.J., Goldsbrough, P.B., and Cobbett, C.S. (1999).

Phytochelatin synthase genes from Arabidopsis and the yeast Schiz-

osaccharomyces pombe. Plant Cell 11: 1153–1164.

Hart, B.A., Lee, C.H., Shukla, G.S., Shukla, A., Osier, M., Eneman, J.

D., and Chiu, J.F. (1999). Characterization of cadmium-induced

apoptosis in rat lung epithelial cells: evidence for the participation of

oxidant stress. Toxicology 133: 43–58.

Herbette, S., et al. (2006). Genome-wide transcriptome profiling of the

early cadmium response of Arabidopsis roots and shoots. Biochimie

88: 1751–1765.

Imura, N., Naganuma, A., and Satoh, M. (1989). Metallothionein as

a resistance factor for antitumor drugs. Gan To Kagaku Ryoho 16:

599–604.

Jarup, L., Berglund, M., Elinder, C.G., Nordberg, G., and Vahter, M.

(1998). Health effects of cadmium exposure–A review of the literature

and a risk estimate. Scand. J. Work Environ. Health 24 (Suppl. 1): 1–51.

Jeyaprakash, A., Welch, J.W., and Fogel, S. (1991). Multicopy CUP1

plasmids enhance cadmium and copper resistance levels in yeast.

Mol. Gen. Genet. 225: 363–368.

Jin, S., Cheng, Y., Guan, Q., Liu, D., Takano, T., and Liu, S. (2006). A

metallothionein-like protein of rice (rgMT) functions in E. coli and its

gene expression is induced by abiotic stresses. Biotechnol. Lett. 28:

1749–1753.

Kim, D.Y., Bovet, L., Maeshima, M., Martinoia, E., and Lee, Y. (2007).

The ABC transporter AtPDR8 is a cadmium extrusion pump conferring

heavy metal resistance. Plant J. 50: 207–218.

Kim, Y.Y., Kim, D.Y., Shim, D., Song, W.Y., Lee, J., Schroeder, J.I.,

Kim, S., Moran, N., and Lee, Y. (2008). Expression of the novel wheat

gene TM20 confers enhanced cadmium tolerance to bakers’ yeast. J.

Biol. Chem. 283: 15893–15902.

Kotak, S., Port, M., Ganguli, A., Bicker, F., and von Koskull-Doring,

P. (2004). Characterization of C-terminal domains of Arabidopsis heat

stress transcription factors (Hsfs) and identification of a new signature

combination of plant class A Hsfs with AHA and NES motifs essential

for activator function and intracellular localization. Plant J. 39: 98–112.

Kotak, S., Vierling, E., Baumlein, H., and von Koskull-Doring, P.

(2007). A novel transcriptional cascade regulating expression of heat

stress proteins during seed development of Arabidopsis. Plant Cell

19: 182–195.

Langmade, S.J., Ravindra, R., Daniels, P.J., and Andrews, G.K.

(2000). The transcription factor MTF-1 mediates metal regulation of

the mouse ZnT1 gene. J. Biol. Chem. 275: 34803–34809.

Lee, J., Bae, H., Jeong, J., Lee, J.Y., Yang, Y.Y., Hwang, I.,

Martinoia, E., and Lee, Y. (2003). Functional expression of a bacterial

heavy metal transporter in Arabidopsis enhances resistance to and

decreases uptake of heavy metals. Plant Physiol. 133: 589–596.

Li, Z.S., Lu, Y.P., Zhen, R.G., Szczypka, M., Thiele, D.J., and Rea, P.

A. (1997). A new pathway for vacuolar cadmium sequestration in


Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathio-

nato)cadmium. Proc. Natl. Acad. Sci. USA 94: 42–47.

Miller, G., and Mittler, R. (2006). Could heat shock transcription factors

function as hydrogen peroxide sensors in plants? Ann. Bot. (Lond.)

98: 279–288.

Mishra, S.K., Tripp, J., Winkelhaus, S., Tschiersch, B., Theres, K.,

Nover, L., and Scharf, K.D. (2002). In the complex family of heat

stress transcription factors, HsfA1 has a unique role as master

regulator of thermotolerance in tomato. Genes Dev. 16: 1555–1567.

Nakai, A. (1999). New aspects in the vertebrate heat shock factor

system: Hsf3 and Hsf4. Cell Stress Chaperones 4: 86–93.

Nover, L., Bharti, K., Doring, P., Mishra, S.K., Ganguli, A., and

Scharf, K.D. (2001). Arabidopsis and the heat stress transcription

factor world: how many heat stress transcription factors do we need?

Cell Stress Chaperones 6: 177–189.

Ogawa, D., Yamaguchi, K., and Nishiuchi, T. (2007). High-level

overexpression of the Arabidopsis HsfA2 gene confers not only

increased themotolerance but also salt/osmotic stress tolerance

and enhanced callus growth. J. Exp. Bot. 58: 3373–3383.

Ortiz, D.F., Ruscitti, T., McCue, K.F., and Ow, D.W. (1995). Transport

of metal-binding peptides by HMT1, a fission yeast ABC-type vacu-

olar membrane protein. J. Biol. Chem. 270: 4721–4728.

Preveral, S., Gayet, L., Moldes, C., Hoffmann, J., Mounicou, S.,

Gruet, A., Reynaud, F., Lobinski, R., Verbavatz, J.M., Vavasseur,

A., and Forestier, C. (2009). A common highly conserved cadmium

detoxification mechanism from bacteria to humans: heavy metal

tolerance conferred by the ATP-binding cassette (ABC) transporter

SpHMT1 requires glutathione but not metal-chelating phytochelatin

peptides. J. Biol. Chem. 284: 4936–4943.

Qi, X., Zhang, Y., and Chai, T. (2007). Characterization of a novel plant

promoter specifically induced by heavy metal and identification of

the promoter regions conferring heavy metal responsiveness. Plant

Physiol. 143: 50–59.

Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Labo-

ratory Manual, 3rd ed. (Cold Spring Harbor, NY: Cold Spring Harbor

Laboratory Press).

Sandalio, L.M., Dalurzo, H.C., Gomez, M., Romero-Puertas, M.C.,

and del Rio, L.A. (2001). Cadmium-induced changes in the growth

and oxidative metabolism of pea plants. J. Exp. Bot. 52: 2115–2126.

Schutzendubel, A., Schwanz, P., Teichmann, T., Gross, K.,

Langenfeld-Heyser, R., Godbold, D.L., and Polle, A. (2001).

Cadmium-induced changes in antioxidative systems, hydrogen per-

oxide content, and differentiation in Scots pine roots. Plant Physiol.

127: 887–898.

Sewell, A.K., Yokoya, F., Yu, W., Miyagawa, T., Murayama, T., and

Winge, D.R. (1995). Mutated yeast heat shock transcription factor

exhibits elevated basal transcriptional activation and confers metal

resistance. J. Biol. Chem. 270: 25079–25086.

Tang, W., Charles, T.M., and Newton, R.J. (2005). Overexpression of

the pepper transcription factor CaPF1 in transgenic Virginia pine

(Pinus Virginiana Mill.) confers multiple stress tolerance and enhances

organ growth. Plant Mol. Biol. 59: 603–617.

Vido, K., Spector, D., Lagniel, G., Lopez, S., Toledano, M.B., and

Labarre, J. (2001). A proteome analysis of the cadmium response in

Saccharomyces cerevisiae. J. Biol. Chem. 276: 8469–8474.

von Koskull-Doring, P., Scharf, K.D., and Nover, L. (2007). The

diversity of plant heat stress transcription factors. Trends Plant Sci.

12: 452–457.

Weber, M., Trampczynska, A., and Clemens, S. (2006). Comparative

transcriptome analysis of toxic metal responses in Arabidopsis

thaliana and the Cd(2+)-hypertolerant facultative metallophyte Arabi-

dopsis halleri. Plant Cell Environ. 29: 950–963.

Wimmer, U., Wang, Y., Georgiev, O., and Schaffner, W. (2005). Two

major branches of anti-cadmium defense in the mouse: MTF-1/

metallothioneins and glutathione. Nucleic Acids Res. 33: 5715–

5727.

Wu, C. (1995). Heat shock transcription factors: structure and regula-

tion. Annu. Rev. Cell Dev. Biol. 11: 441–469.

Yamanouchi, U., Yano, M., Lin, H., Ashikari, M., and Yamada, K.

(2002). A rice spotted leaf gene, Spl7, encodes a heat stress tran-

scription factor protein. Proc. Natl. Acad. Sci. USA 99: 7530–7535.

Yepiskoposyan, H., Egli, D., Fergestad, T., Selvaraj, A., Treiber, C.,

Multhaup, G., Georgiev, O., and Schaffner, W. (2006). Transcrip-

tome response to heavy metal stress in Drosophila reveals a new zinc

transporter that confers resistance to zinc. Nucleic Acids Res. 34:

4866–4877.

Yokotani, N., Ichikawa, T., Kondou, Y., Matsui, M., Hirochika, H.,

Iwabuchi, M., and Oda, K. (2008). Expression of rice heat stress

transcription factor OsHsfA2e enhances tolerance to environmental

stresses in transgenic Arabidopsis. Planta 227: 957–967.

Zhang, B., Georgiev, O., Hagmann, M., Gunes, C., Cramer, M.,

Faller, P., Vasak, M., and Schaffner, W. (2003). Activity of metal-

responsive transcription factor 1 by toxic heavy metals and H2O2

in vitro is modulated by metallothionein. Mol. Cell. Biol. 23: 8471–

8485.

Zhou, G., Xu, Y., Li, J., Yang, L., and Liu, J.Y. (2006). Molecular

analyses of the metallothionein gene family in rice (Oryza sativa L.). J.

Biochem. Mol. Biol. 39: 595–606.

Zhou, J., and Goldsbrough, P.B. (1994). Functional homologs of fungal

metallothionein genes from Arabidopsis. Plant Cell 6: 875–884.


DOI 10.1105/tpc.109.066902; originally published online December 22, 2009;Plant Cell

Martinoia and Youngsook LeeDonghwan Shim, Jae-Ung Hwang, Joohyun Lee, Sichul Lee, Yunjung Choi, Gynheung An, Enrico

in Wheat and RiceOrthologs of the Class A4 Heat Shock Transcription Factor HsfA4a Confer Cadmium Tolerance

This information is current as of November 24, 2020

Supplemental Data /content/suppl/2009/12/02/tpc.109.066902.DC1.html

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

orthologs of the class a4 heat shock transcription …...2009/12/22 · orthologs of the class a4...

Documents