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. 2009 Dec;21(12):4031–4043. doi: 10.1105/tpc.109.066902

Orthologs of the Class A4 Heat Shock Transcription Factor HsfA4a 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, Youngsook Lee a,2
PMCID: PMC2814514  PMID: 20028842

Abstract

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 damage by displacing cofactors from a variety of proteins, including transcription 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 shares many 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 synthesize 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. Sequestration 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 Saccharomyces 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 vacuole (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 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 metallothionein (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 conserved 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 expression (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 a heat shock transcription factor of class A4 (Ta HsfA4a) 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 (Δycf1) with a wheat root cDNA library. Cd sequestration into the vacuole is defective in Δycf1; thus, this strain does not grow on half-strength SG agar plates containing 60 to 80 μM CdCl2. We selected colonies that grew in the presence of 60 to 80 μM 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 Transmembrane 20 (TM20) (Kim et al., 2008). The third encoded a 432–amino acid polypeptide with homology to heat shock transcription factors. We named the encoded protein Ta HsfA4a (GenBank accession number FJ790791), according to the nomenclature proposed by Nover et al. (2001). Ta HsfA4a contains a putative HSF 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 characterized 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).

Δycf1 cells expressing Ta HsfA4a exhibited dramatically enhanced growth when compared with Δycf1 cells transformed with the empty vector (EV) on half-strength SG agar medium supplemented with 40 μM CdCl2 (Figure 1). The growth of Δycf1 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 tolerance to the other heavy metals, namely, lead, zinc, cobalt, manganese, silver, mercury, or iron (see Supplemental Figure 2 online). The cellular localization of Ta HsfA4a was 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 Commelina communis guard cells that transiently expressed the construct after biolistic bombardment (see Supplemental Figure 3B online).

Figure 1.

Figure 1.

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Enhanced Cd Tolerance by HsfA4a in Yeast.

(A) Expression of Ta HsfA4a–conferred Cd tolerance in the Δycf1 yeast mutant. Enhanced growth of Ta HsfA4a–transformed yeast on half-strength SG-ura agar plates supplemented with 40 μM 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 μM CdCl2. The data are means and se from three independent experiments.

Os HsfA4a Is a Rice Ortholog of Ta HsfA4a

We searched the plant EST database for homologs of Ta HsfA4a. As shown in the phylogenetic tree presented in Figure 2A, the amino acid sequence of Ta HsfA4a 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).

Figure 2.

Figure 2.

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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 Δycf1 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.]

We investigated whether Ta HsfA4a homologs from rice and Arabidopsis confer Cd tolerance in the Δycf1 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 (Figure 2B). The transformation of yeast cells with the three Arabidopsis 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).

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). We generated several chimeric proteins of Ta HsfA4a and Os HsfA4d and tested their promotion of Cd tolerance in yeast (Figure 3). 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 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 Δycf1 (Figure 3B). These results suggest that the DBD of Ta HsfA4a is critical for Cd resistance.

Figure 3.

Figure 3.

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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 Δycf1 yeast cells transformed with domain-swapped mutants on half-strength SG-ura agar plates supplemented with 40 μM CdCl2. SwapI alone exhibited Cd resistance comparable with that of Ta HsfA4a. SwapII and SwapIII did not improve Cd tolerance significantly.

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 region were different in Ta HsfA4a and Os HsfA4a when compared with the other Hsfs (Figures 4A and 4B). The Ser and Trp amino acid residues, which are 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 Δycf1 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.

Figure 4.

Figure 4.

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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 μM CdCl2. The double mutation (S39A and W50L) significantly enhanced the growth of Δycf1, compared with either of the S39A or W50L single mutations. The data are means and se from three independent experiments.

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 the many 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 Supplemental Table 1 online). The individual expression of these six genes in yeast cells revealed that only CUP1 (which encodes metallothionein) improved Cd tolerance substantially. CUP1 was 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 the CUP1 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 Δcup1 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 CdCl2 at concentrations as low as 3 μM. Ta HsfA4a overexpression in this mutant did not enhance Cd tolerance of the yeast cells, which remained sensitive to 3 μM CdCl2 (Figure 5B). By contrast, expression of Ta HsfA4a in the CUP1 single-copy mutant (cup1s) caused an increase in the level of CUP1 expression and enabled the cup1s strain to grow on 3 μM 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.

Figure 5.

Figure 5.

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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 (Δcup1) on half-strength SG-ura agar plates supplemented with 3 μM CdCl2.

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 overexpressing Ta HsfA4a (Ta HsfA4a OX1 and OX2) was compared on half-strength Murashige and Skoog (MS) agar supplemented with 0, 100, 200, or 300 μM 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 in media containing Cd (100, 200, or 300 μM) (Figure 6A, right panels; see quantification in Figure 6B). These results demonstrate that Ta HsfA4a confers Cd tolerance in rice. We also tested whether Ta HsfA4a 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).

Figure 6.

Figure 6.

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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 μM 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 μM CdCl2. Bars = 3 cm.

(B) Quantitative measurement of the height of Ta HsfA4a–overexpressing plants (mean ± 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 ± sd).

Next, we wondered whether HsfA4a also upregulates metallothionein in rice, as it did in yeast. Among the 11 metallothioneins described in rice, we speculated that MT-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 transformed 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 in medium containing 150 μM CdCl2 (Figure 7; see Supplemental Figure 7 online). The first allele (hsfA4a-1, 3A-13297) had a T-DNA insertion in the promoter region (−189 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.

Figure 7.

Figure 7.

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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 ± se).

(C) Photographs of representative wild-type and hsfA4a-1 knockdown plants in the presence and absence of 150 μM 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 ± se, n = 36).

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 μM 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 truncated mutant protein Os HsfA4a (Δ106 amino acids) conferred Cd tolerance to Δycf1 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). Taken together, our results demonstrate that HsfA4a indeed plays an important role in Cd tolerance in rice.

Cd Induces MT Expression in Rice and Wheat

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 seedlings were exposed to 100 μM CdCl2 for 2 h, and the transcript levels of target genes were analyzed using quantitative real-time PCR (Figure 8). Cd treatment increased Os HsfA4a expression by more than fourfold in roots of rice plants, compared with expression 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 Os MT-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 increases in the transcript levels of Ta HsfA4a, Ta MT-I-1, and Ta MT-I-2 in the roots (Figure 8B). These results show that HsfA4a 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.

Figure 8.

Figure 8.

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Induction of HsfA4a and MT Genes 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 μM 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 ΔΔCT. G3PDH and Act1 were analyzed as a constitutively expressed control gene in wheat and rice, respectively. Data represent three independent experiments (mean ± se, n = 9).

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 (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 hypersensitivity in rice plants. We propose that HsfA4a functions in Cd tolerance in planta because HsfA4a genes are upregulated by Cd 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–induced MT expression is crucial for Ta HsfA4a–mediated Cd tolerance in yeast. Metallothionein 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 induced CUP1 expression in yeast and Os MT-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, Os MT-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.

Although metallothionein 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, unpublished 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 tolerance mechanisms may 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 Supplemental 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, unpublished data). An intriguing question remains regarding the mechanisms underlying the Cd specificity of the HsfA4a-induced response. HsfA4a might activate target genes that are specifically 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 heavy metals (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 and Os HsfA4a conferred Cd tolerance to yeast and plants, but other Hsfs with 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 tolerance 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 transcription factor in animal cells, we found HsfA4a orthologs as Cd tolerance genes in wheat and rice plants. Putative metal-responsive 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 to MTF-1. In plant, unique metal-responsive transcription factor(s) that are structurally distinct 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 factors, including Hsfs, are upregulated by Cd 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 because we could not find any dicot genes that are highly homologous to Ta HsfA4a in their DBDs (Figure 2A).

In conclusion, we have demonstrated the function of two orthologs of the plant class A4 Hsfs, 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 sativa Dongjin), 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 containing 1.5% sucrose, 0.8% phytoagar, and 0.28 mM myoinositol (Sigma-Aldrich), in the presence or absence of 100 to 300 μM CdCl2, 100 μM CuCl2, 3 mM Pb(NO3)2, and 5 mM ZnCl2. Seedlings were grown for 7 d at 27°C under continuous light and were then transferred to soil in a greenhouse and raised to maturity.

Isolation of Cd Tolerance Genes from a Wheat 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 (MATα ura3-52 his6 leu2-3,-112 his3-Δ200 trp1-901 lys2-801 suc2-Δ, ycf1∷hisG) using the lithium acetate method (Ito et al., 1983). Colonies that grew on medium containing 60 to 80 μM CdCl2 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 Δycf1 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), the middle fragment containing HR-A/B and NLS (P2), or the C-terminal fragment containing AHA and NES (P3) were synthesized by PCR from plasmid 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 synthesized using Os HsfA4d_F1 and Os 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 and Os 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 second PCR from the mix of P1 and P6 using Ta HsfA4a_F1 and Os HsfA4d_R3. The Swap III DNA was 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, we used site-directed mutagenesis to generate the amino acid substitutions in the DBD of Os HsfA4d (S39A and W50L) (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 Δycf1, cup1s (MATα, trp1-1, leu2-3, leu2-112, gal1, ura3-50, His-, cup1 single copy), and Δcup1 (MATα, 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 acetate method. Transformants were selected on minimal medium lacking uracil. To perform the Cd 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 μM CdCl2 at 30°C 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 galactose-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 and MTs in wheat and rice plants, total RNA (3 μg) was subjected to cDNA synthesis using a Powerscript RT kit (Clontech) according to the manufacturer's instructions. Quantitative real-time PCR was performed 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 dehydrogenase (G3PDH) was used as an internal control in PCR amplification, 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 and Os MT-I-1a_RT_R, Ta MT-I-1_F and Ta MT-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 ΔΔCT. 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); Glycine max (Gm) HsfA4a (TC135284); Hordeum vulgare (Hv) HsfA4a (TC42448); Lycopersicon esculentum (Le) HsfA4a (BT014619) and HsfA4b (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 nomenclature 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 Δycf1 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 Δycf1 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.

Supplementary Material

[Supplemental Data]
tpc.109.066902_index.html (835B, html)

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.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Youngsook Lee (ylee@postech.ac.kr).

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Some figures in this article are displayed in color online but in black and white in the print edition.

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Online version contains Web-only data.

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