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Keywords:

  • Arabidopsis;
  • environmental stress;
  • heat-shock transcription factor;
  • hydrogen peroxide;
  • microarray;
  • subtractive hybridization

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We isolated 76 high-light and heat-shock (HL + HS) stress-inducible genes, including a putative heat-shock transcription factor (HsfA2), by suppression-subtractive hybridization from Arabidopsis. The transcript level of HsfA2 was significantly increased under the several stress conditions or by the H2O2 treatment. Furthermore, the induction of HsfA2 expression was highest among those of other class A HSFs in response to HL + HS stress conditions. The promoter assay revealed that HsfA2 is induced mainly in rosette leaves under HL + HS stress conditions. In the HsfA2-overexpressing Arabidopsis (Pro35S:HsfA2) plants, 46 genes, including a large number of heat-shock proteins, ascorbate peroxidase 2 and galactinol synthase 1 and 2, were highly expressed compared with those in the wild-type plants. The transcript levels of the HsfA2 target genes are highly correlated with those of HsfA2 in the Pro35S:HsfA2 plants. The transcript levels of the HsfA2 target genes, as well as HsfA2 transcripts, were induced by treating with exogenous H2O2. In the knockout HsfA2 Arabidopsis plants, the induction of 26 HsfA2 target genes was strongly reduced for up to 2 h under HL + HS stress conditions. Furthermore, the Pro35S:HsfA2 plants showed increased tolerance to combined environmental stresses. Our present results indicate that HsfA2 is a key regulator in the induction of the defence system under several types of environmental stress.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

With the completion of the Arabidopsis genome sequence, the entire complement of genes coding for transcription factors has been identified and described (Riechmann et al., 2000). The Arabidopsis genome coded for at least 1533 transcriptional regulator genes, more than that of Drosophila and Caenorhabditis elegans (Riechmann et al., 2000). Furthermore, higher plants possess plant-specific transcription factors, the WRKY and NAC transcription families. This appears to result from the fact that plants, unlike animals, are unable to move and therefore experience a wide array of environmental stresses, such as high-light (HL), drought, high temperature, chilling, salinity and air pollution. Accordingly, plants have developed complicated defence mechanisms in response to various types of environmental stress.

Many researchers have studied the defence mechanisms and their signal transduction regulation systems in higher plants under various types of environmental stress (Kimura et al., 2003; Kreps et al., 2002; Rossel et al., 2002; Seki et al., 2001, 2002). Recently, it has become clear that the response of plant cells encountering a single stress condition is different from that under conditions in the field, where a number of different stresses may occur simultaneously. Changes in steady-state levels of transcripts were examined in the leaves of tobacco or Arabidopsis plants that were subjected to drought or heat-shock (HS) stresses, or a combination of these (Rizhsky et al., 2002, 2004a). Although a small number of transcripts were specifically expressed during the combination of drought and HS stresses, there was a considerable degree of overlap between the transcripts expressed in plants during drought or HS stresses, and a combination of drought and HS stresses. These observations indicated that the transcription factors commonly induced by several environmental stresses are important in inducing defence systems against different types of environmental stress. Here, to characterize the defence systems against a wide range of environmental stresses in plants, we first isolated a combination of HL and HS stress-inducible genes from Arabidopsis using suppression-subtractive hybridization (SSH), and explored the H2O2-induced transcription factors among them. As a result, a putative heat-shock transcription factor (HSF), HsfA2, was isolated.

There are a total of four HSFs in vertebrates, only one HSF in Drosophila and C. elegans, and one HSF plus three HSF-related proteins in yeast (Nakai, 1999; Nover et al., 1996). In contrast, 21 open reading frames encoding putative HSFs were identified in the Arabidopsis genome (Nover et al., 2001). A minimum of 23 HSF genes were identified in the available parts of the rice genome (Kotak et al., 2004). Search of expressed sequence tag databases indicated the presence of at least 18 HSFs in tomato and 34 in soybean. Based on the presence of the conserved DNA-binding domain plus the adjacent oligomerization domains (HR-A/B region), plant HSFs are assigned to classes A–C (Nover et al., 2001). Short peptide motifs enriched with aromatic and large hydrophobic amino acid residues embedded in an acidic surrounding (AHA motifs) are essential for the activity of class A HSFs. In contrast, class B and C HSFs lack AHA motifs and have no activator function of their own (Kotak et al., 2004).

It has been reported that HSF controls the expression of heat-shock protein (HSP) genes and functions as a transcriptional activator (Kotak et al., 2004; Prändl et al., 1998; Schöffl et al., 1998). The tomato HsfA1 (LpHsfA1) protein was localized in the nucleus under HS stress conditions, whereas most of the LpHsfA2 proteins were found in the cytoplasm under the same conditions (Lyck et al., 1997; Scharf et al., 1998). Interestingly, localization of the LpHsfA2 protein to the nucleus evidently required interaction with LpHsfA1 (Scharf et al., 1998). However, Arabidopsis HsfA2 protein as a transcriptional activator can localize to the nucleus without interacting with the HsfA1 protein (Kotak et al., 2004). Lohmann et al. (2004) have reported that HsfA1a and HsfA1b are key regulators of the immediate stress-induced activation of HS gene transcription, including Hsp83.1 and Hsp17.6. The HsfA1a and HsfA1b double-knockout Arabidopsis mutant (KO-HsfA1a/1b) was markedly impaired in the early and transient mRNA accumulation of some HSP genes during the first hour of HS treatment (Lohmann et al., 2004). Similar results have been reported from transgenic Arabidopsis plants that overexpressed the dominant-negative HsfA1a protein (EN-HSF1) (Wunderlich et al., 2003). However, after 2 h of HS, the differences in the transcript levels of Hsp83.1 and Hsp17.6 between KO-HsfA1a/1b and wild-type plants become almost negligible (Lohmann et al., 2004). Furthermore, expressions of HsfA2, HsfA4a and HsfB2b in wild-type plants showed similar levels in KO-HsfA1a/1b plants (Busch et al., 2005), indicating that HsfA2 is not the target gene of HsfA1a/1b. As the genetic redundancy of HSFs in plants complicates the analysis of their individual roles, little is known about the individual role of HSFs as a transcriptional factor, including HsfA2.

By the transcriptome analysis using HsfA2-overexpressing (Pro35S:HsfA2) and -knock out (KO-HsfA2) Arabidopsis plants, it has been demonstrated that transcript levels of 46 genes involved in the stress defence were directly or indirectly regulated by HsfA2. Furthermore, the Pro35S:HsfA2 plants showed increased tolerance to combined environmental stresses. Our findings presented here indicate that HsfA2 plays an important role in regulating induction of the defence system in response to different types of environmental stress.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Isolation of genes responsive to a combination of HL and HS stress using SSH

Genes responsive to a combination of HL + HS stress were captured through the construction of a subtractive cDNA library from mRNAs isolated from 2-week-old Arabidopsis plants exposed to a combination of HL + HS stress (800 μE m−2 sec−1, 40°C) for 1 h. The subtracted cDNA clones (768 clones) were isolated and screened with reverse Northern analysis, then 611 positive clones were sequenced. The sequences of clones were evaluated by comparing them with the nucleotide sequences deposited in National Center for Biotechnology Information (NCBI) databases using the blast program, and as a result 76 genes were identified (Table S1 in Supplementary Material). All the HL + HS stress-inducible genes, including a number of genes encoding HSPs, were functionally classified. Two genes encoding active oxygen-scavenging enzymes, glutathione S-transferase 6 (GST6) and peroxiredoxin type 2 B (Prx II B), were also induced under HL + HS stress conditions.

Furthermore, HL + HS stress-inducible genes included three transcription factors encoding the DREB2A, putative HSF (HsfA2) and NAC domain (petunia NAM and Arabidopsis ATAF1, ATAF2 and CUC2)-containing protein.

Expression of HsfA2 in response to several stress conditions

To explore the gene-regulation system of HsfA2, the expression of HsfA2 in response to several stress conditions was analysed using RNA gel-blot analysis. The transcript level of HsfA2 markedly increased within 15 min and reached a peak after 45 min under the HL + HS stress conditions (Figure 1a). HsfA2 was also rapidly induced by HS stresses only (dark, 45°C). In addition, the HL stress at a normal temperature (800 μE m−2 sec−1, 25°C) increased the transcript level of HsfA2. Interestingly, the transcript level of HsfA2 was also slightly increased by chilling stresses (CH; 100 μE m−2 sec−1, 4°C). Furthermore, the transcript level of HsfA2 was increased by the addition of exogenous H2O2 (10 mm) within 1 h in Arabidopsis T87 cells under low light at 100 μE m−2 sec−1 (Figure 1b), suggesting that the increase in the H2O2 level in plant cells mediates the expression of HsfA2. The addition of abscisic acid (ABA; 100 μm) to T87 cell cultures did not increase the transcripts ofHsfA2.

image

Figure 1.  Effect of several stresses and treatments with endogenous H2O2 or abscisic acid (ABA) on transcript levels of HsfA2. (a) Two-week-old Arabidopsis plants (control, 100 μE m−2 sec−1, 25°C) were transferred to a combination of high-light and heat-shock (HL + HS, 800 μE m−2 sec−1, 40°C), heat-shock (HS, dark, 45°C), high-light (HL, 800 μE m−2 sec−1, 25°C) or chilling (CH, 100 μE m−2 sec−1, 4°C) stress conditions. Transcript levels of HsfA2 were detected using RNA gel-blot analysis as described in Experimental procedures. Each lane was loaded with 20 μg total RNA. Equal loading was verified by rRNA visualization after ethidium bromide staining. (b) Three-day-old Arabidopsis T87 cells were untreated (control, 100 μE m−2 sec−1, 22°C) or treated with 10 mm H2O2 or 100 μm ABA, under light conditions (100 μE m−2 sec−1, 22°C).

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Expression of class A HSF genes in response to HL and HS stress

In Arabidopsis, 15 genes are identified as homologues of class A HSF (Nover et al., 2001). In order to determine whether HsfA2 is a key regulator of the HL + HS stress response, we analysed the expression profiles of all class A HSF genes using quantitative PCR analysis under HL + HS stress conditions (Figure 2). The transcript levels of HsfA2, HsfA7a and HsfA7b were markedly increased during HL + HS stress conditions. In particular, the induction level of HsfA2 expression was higher than those of HsfA7a and HsfA7b at 60 min under the HL + HS stress conditions. Therefore, it seems likely that the HsfA2 protein, among other class A HSFs, plays a most important role in the induction of defence systems in response to these environmental stresses.

image

Figure 2.  Expression profile of Arabidopsis class A HSF genes under high-light and heat-shock (HL + HS) stress conditions. Two-week-old wild-type plants were transferred to a combination of HL + HS stress conditions. Total RNA was reverse-transcribed into cDNA and used as a template for quantitative reverse transcriptase-polymerase chain reaction as described in Experimental procedures. Specific primers for class A HSFs (HsfA1a; A1b; A1d; A1e; A2; A3; A4a; A4c; A5; A6a; A6b; A7a; A7b; A8; A9) and Actin2 standard were designed. Relative transcript levels were normalized to Actin2mRNA (=100%). Data are mean values ± SD of three individual experiments (n = 3).

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Tissue-specific expression of HsfA2 gene

To analyse the tissue-specific expression of the HsfA2 gene, we constructed a chimeric gene consisting of the HsfA2 promoter fused to a β-glucuronidase (GUS) reporter gene (pBI/Pro.HsfA2:GUS). The HsfA2 promoter included the sequence from 2 kbp upstream to 174 bp downstream of the site of initiation of transcription. Two-week-old wild-type and transgenic Arabidopsis (ProHsfA2:GUS) plants were transferred to the HL + HS stress conditions. RNA gel-blot analysis revealed that the transcript level of the GUS gene under the control of the HsfA2 promoter, as well as that of endogenous HsfA2 transcripts, was clearly increased under HL + HS stress conditions (Figure 3a).

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Figure 3.  Tissue-specific expression of HsfA2 in Arabidopsis plants. Two-week-old transgenic Arabidopsis plants (control) containing the HsfA2 promoter fused to the GUS reporter gene (ProHsfA2:GUS) were transferred to high-light and heat-shock (HL + HS) stress conditions for 3 h. (a) RNA gel-blot analysis of HsfA2 and GUS genes in transgenic Arabidopsis plants. The transcripts HsfA2 and GUS were detected using RNA gel-blot analysis, as described in Experimental procedures. Each lane was loaded with 20 μg total RNA. (b–f) Histochemical analysis of GUS activity in ProHsfA2:GUS plants. Two-week-old ProHsfA2:GUS plants (b) were transferred to HL + HS stress conditions (c). (d) Leaf of ProHsfA2:GUS plants under HL + HS stress conditions; (e) root tip of ProHsfA2:GUS plants under HL + HS stress conditions; (f) root branch points of ProHsfA2:GUS plants under HL + HS stress conditions.

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GUS activity was not detected in rosette leaves of transgenic Arabidopsis plants grown under normal conditions, while it was detected in transgenic plants exposed to the HL + HS stress conditions (Figure 3b–d). Additionally, GUS activity was strongly observed in veins, root tips and root branch points (Figure 3e and f).

Identification of target genes of HsfA2

To identify the target genes of HsfA2, we generated transgenic Arabidopsis plants overexpressing HsfA2 (14 lines) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The transcript levels of HsfA2 in the transgenic Arabidopsis plants (Pro35S:HsfA2) were examined by RNA gel-blot analysis using HsfA2 cDNA as a probe. All the T3 transformants showed overexpression of the transgenes (data not shown). Thus we selected three HsfA2 lines for further analysis. In the Pro35S:HsfA2–3, -6, and -12 plants, the levels of the HsfA2 transcript were approximately 20-, 80- and 30-fold higher, respectively, than those of the wild-type plants (Figure 4). The transcript levels of 76 HL + HS stress-inducible genes, which were isolated with SSH in the HL + HS stressed plants, were analysed with reverse Northern analysis in the Pro35S:HsfA2–6 plants. Not only HsfA2, but also 15 HL + HS stress-inducible genes, were markedly induced in the Pro35S:HsfA2–6 plants compared with those in the wild-type plants under normal conditions (data not shown). The transcript levels of the 16 HL + HS stress-inducible genes, including HsfA2, were also examined using RNA gel-blot analysis (Table 1; Figure 4). The same genes were induced in the Pro35S:HsfA2–3 and -12 plants. The transcript levels of those genes correlated positively with those of HsfA2 in the each transgenic line (Figure 4). These findings indicate that overexpression of the HsfA2 protein specifically induces expression of the 15 genes designated as putative HsfA2 target genes.

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Figure 4.  Expression of putative HsfA2 target genes in wild-type and HsfA2-overexpressing plants. Total RNAs were isolated from wild-type and Pro35S:HsfA2 plants grown for 2 weeks under normal conditions. Transcript levels of HsfA2 and HsfA2 target genes were detected using RNA gel-blot analysis, as described in Experimental procedures. Each lane was loaded with 20 μg total RNA. In Pro35S:HsfA2–3, -6, and -12 plants, levels of the HsfA2 transcript were approximately 20-, 80- and 30-fold higher, respectively, than those of wild-type plants.

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Table 1.    Upregulated genes in HsfA2 target genes in Pro35S:HsfA2–6 plants compared with wild-type plants under normal conditions
AGI No.AnnotationReverse NorthernMicroarray
  1. ×, identification method.

Cell rescue, defence and virulence
 At1g5256026.5-kDa class I small heat-shock protein (HSP)-like (Hsp26.5-P)××
 At1g5405017.4-kDa class III HSP (Hsp17.4-CIII)× 
 At1g74310HSP 101 (Hsp101)× 
 At2g2950017.6-kDa class I small HSP (Hsp17.6B-CI)××
 At2g32120HSP 70 family protein (Hsp70T-2)× 
 At3g12580HSP 70, putative (Hsp70)× 
 At4g2767025.3-kDa small HSP, chloroplast precursor (Hsp25.3-P)××
 At5g1203017.7-kDa class II HSP 17.6A (Hsp17.7-CII)××
 At5g5972018.1-kDa class I HSP (Hsp18.1-CI)××
 At4g1025022.0-kDa ER small HSP (Hsp22.0-ER) ×
 At1g5354017.6-kDa class I small HSP (Hsp17.6C-CI) ×
 At1g16030HSP 70, putative (Hsp70B) ×
 At3g09640l-ascorbate peroxidase 2 (APX2) ×
 At3g04720Hevein-like protein (HEL) ×
 At4g2520023.6-kDa mitochondrial small HSP (Hsp23.6-M) ×
 At5g15970Cold-regulated protein COR6.6 (KIN2) ×
 At5g3767015.7-kDa class I-related small HSP-like (Hsp15.7-CI) ×
 At2g42540Cold-regulated protein cor15a precursor ×
Metabolism
 At2g22240Inositol-3-phosphate synthase isozyme 2 (IPS2)××
 At2g47180Galactinol synthase (GolS1)× 
 At1g56600Galactinol synthase2 (GolS2) ×
 At4g37800Hydrolase, acting on glycosyl bonds/hydrolase, hydrolysing O-glycosyl compounds ×
Transcription
 At3g16770AP2 domain-containing protein RAP2.3 (RAP2.3) ×
Cellular transport, transport facilitation and transport routes
 At3g26540Pentatricopeptide repeat-containing protein ×
 At3g10780emp24/gp25L/p24 family protein ×
 At5g56290Peroxisomal targeting signal type 1 receptor (PEX5) ×
 At5g59310Lipid transfer protein 4 (LTP4) ×
Cell fate
 At5g13170Nodulin MtN3 family protein ×
Protein fate (folding, modification, destination)   
 At5g46740Ubiquitin-specific protease 21 (UBP21) ×
Cellular communication/ signal transduction mechanism
 At2g41090Calmodulin-like calcium-binding protein, 22 kDa (CaBP-22) ×
Unclassified protein
 At4g12400Stress-inducible protein (sti1), putative× 
 At2g46240IQ domain-containing protein/ BAG domain-containing protein× 
 At4g28820Zinc finger (HIT-type) family protein ×
 At3g58760Ankyrin-kinase, putative ×
 At3g03270Universal stress protein family protein ×
 At2g05440Putative glycine-rich protein ×
 At5g15160bHLH family protein ×
 At5g42780Zinc finger homeobox family protein/ ZF-HD homeobox family protein ×
Unknown protein
 At1g17870Expressed protein× 
 At2g19310Expressed protein× 
 At3g49200Hypothetical protein ×
 At5g19710Hypothetical protein ×
 At1g17710Expressed protein ×
 At4g09890Expressed protein ×
 At5g07330Putative protein ×
 At3g45160Expressed protein ×

In order to identify other putative HsfA2 target genes, we conducted a transcriptome analysis of the Pro35S:HsfA2–6 and wild-type plants using a DNA microarray (Agilent Arabidopsis 22K; Agilent Technologies, Palo Alto, CA, USA). The transcript levels of 37 genes, including some types of HSP, ascorbate peroxidase 2 (APX2), galactinol synthase 2 (GolS2) and inositol 3-phosphate syntase 2 (IPS2), were induced, and those of nine genes were suppressed in the Pro35S:HsfA2–6 plants compared with wild-type plants under normal conditions (Tables S2 and S3). Some of the putative HsfA2 target genes identified by the RNA gel-blot analysis, as described above, were not significantly induced in the DNA microarray analysis in Pro35S:HsfA2–6 plants; we confirmed the induction of these genes in Pro35S:HsfA2 plants using quantitative PCR analysis (data not shown). Thus a total of 46 genes were induced in Pro35S:HsfA2–6 plants compared with wild-type plants under normal conditions (Table 1).

Effect of HL and HS stress on induction of target genes

The expression patterns of not only the HsfA2 gene, but also the putative HsfA2 target genes, were analysed using RNA gel-blot analysis under HL + HS stress conditions. The transcript levels of putativeHsfA2 target genes and HsfA2 were rapidly increased in response to the HL + HS stress conditions (Figure 5a). Furthermore, the transcript levels of the HsfA2 target genes were induced by treating with 10 mm H2O2, as were the transcript levels of HsfA2 in Arabidopsis T87 cells (Figure 5b).

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Figure 5.  Effect of high-light and heat-shock (HL + HS) stresses and treatments with exogenous H2O2 on transcript levels of putative HsfA2 and HsfA2 target genes. (a) Two-week-old Arabidopsis plants (control) were transferred to HL + HS stress conditions. Transcript levels of HsfA2 and HsfA2 target genes were detected using RNA gel-blot analysis, as described in Experimental procedures. Each lane was loaded with 20 μg total RNA. (b) Three-day-old Arabidopsis T87 cells were untreated (control; 100 μE m−2 sec−1, 22°C) or treated with 10 mm H2O2 under light conditions (100 μE m−2 sec−1, 22°C).

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Inhibition of the induction of HsfA2 target genes in knockout HsfA2 plants under HL and HS conditions

Putative heat-shock elements (HSEs) are palindromic HSF-binding motifs, conserved in the promoters of heat shock-inducible genes of all eukaryotes (Bienz and Pelham, 1987; Nover, 1987). The eukaryotic HSE consensus sequence has been defined ultimately as alternating units of 5′-nGAAn-3′. In higher plants, the optimal HSE core consensus was shown to be 5′-aGAAg-3′ (Barros et al., 1992). We searched for the occurrence of perfect and imperfect HSE sequences in consensus with -GAA-/-TTC-, the binding site of HSF, in the putative promoter regions (within 1000 bp upstream of the coding region) of putative 46 HsfA2 target genes (Table S4). Analysis of the promoter region of the HsfA2 target genes revealed that the perfect HSE sequences exist in the putative promoter region of the 21 HsfA2 target genes. Imperfect HSE sequences existed in the regions of all HsfA2 target genes, strongly suggesting that the induction of these genes is regulated by the HsfA2 protein.

To determine the HsfA2 target genes, we obtained an Arabidopsis line containing a T-DNA insert in the second exon of the HsfA2 gene from the SIGnAL project (http://signal.salk.edu/tabout.html). Quantitative PCR analysis revealed that the T-DNA insertion in the knockout HsfA2 (KO-HsfA2) plants resulted in the complete loss of HsfA2 expression at the transcript level under normal or HL + HS stress conditions (Figure 6). The growth and appearance of the KO-HsfA2 plants were similar to those of wild-type plants under normal conditions (data not shown). The transcript levels of the putative 26 HsfA2 target genes were markedly reduced in the KO-HsfA2 plants during HL + HS stress conditions, compared with those of wild-type plants (Figure 6). On the other hand, there was no significant difference in the transcript levels of 12 genes between wild-type and KO-HsfA2 plants under HL + HS stress conditions (data not shown). Furthermore, a slight reduction of the transcript levels of eight genes was observed in KO-HsfA2 under either normal or HL + HS stress conditions (data not shown), suggesting that HsfA2 was not directly associated with the induction of these genes.

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Figure 6.  Effect of high-light and heat-shock (HL + HS) stresses on transcript levels of HsfA2, HsfA1a, HsfA1b and HsfA2 target genes in wild-type and KO-HsfA2 plants. Two-week-old wild-type (bsl00043) and KO-HsfA2 (•) plants were transferred to HL + HS stress conditions. Total RNA was reverse-transcribed into cDNA and used as a template for quantitative reverse transcriptase-polymerase chain reaction, as described in Experimental procedures. Specific primers for HsfA2, HsfA1a, HsfA1b, HsfA2 target genes and Actin2 standard were designed. Relative transcript levels were normalized to Actin2mRNA (=100%). Data are mean values ± SD of three individual experiments (n = 3). Asterisks indicate that mean values are significantly different compared with those in wild-type plants (P < 0.05).

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We observed a slow induction of some HsfA2 target genes under HL + HS stress conditions in the KO-HsfA2 plants (Figure 6), suggesting that other HSFs may mediate slow induction of these genes. Among HsfA2 target genes, 11 genes [Hsp26.5-P;Hsp25.3-P;Hsp18.1-CI;Hsp22.0-ER;Hsp23.6M;Hsp15.7-CI;APX2;GolS1;GolS2; universal stress protein (USP); At1g17870] were identified as HsfA1a/1b target genes (Busch et al., 2005; Panchuk et al., 2002; Wunderlich et al., 2003). To examine whether HsfA1a/1b mediates a slow induction of HsfA2 target genes in the KO-HsfA2 plants under HL + HS stress conditions, the transcript levels of HsfA1a and HsfA1b were investigated under HL + HS stress conditions in wild-type and KO-HsfA2 plants. The transcript levels of HsfA1a and HsfA1b were not increased in both plants under stress conditions (Figure 6), indicating that HsfA1a and HsfA1b are not directly associated with the slow induction of HsfA2 target genes.

Effect of overexpression or absence of HsfA2 on environmental stress tolerance

As the expression of certain genes involved in cellular defence was induced or suppressed in Pro35S:HsfA2 or KO-HsfA2 plants (Tables S1 and S2; Figures 4 and 6), there is a possibility that the Pro35S:HsfA2 and the KO-HsfA2 plants have increased tolerance and high sensitivity, respectively, to several types of environmental stress. Three-week-old Pro35S:HsfA2, KO-HsfA2 and wild-type Arabidopsis plants were exposed to HL, HS or CH stress conditions. However, the absence of the HsfA2 protein did not lead to high sensitivity to these stresses (data not shown). Furthermore, no differences in tolerance to each stress were observed between Pro35S:HsfA2 and wild-type plants (data not shown). As expression of HsfA2 was rapidly induced at a high level under HL, HS or CH stress conditions, it seems likely that there is no effect of additional expression of exogeneous HsfA2 on tolerance to these stresses in the wild-type plants. Accordingly, the Pro35S:HsfA2 plants were subjected to extremely severe stress conditions: HL + HS plus treatment with 50 μm methylviologen (MV) (45°C, 1600 μE m−2 sec−1).

At 3 h after stress treatment, the wilting phenotypes were observed in wild-type and KO-HsfA2 plants, but not in Pro35S:HsfA2–6 plants. The wild-type and KO-HsfA2 plants developed more severe visible leaf injury than the Pro35S:HsfA2–6 plants at 5 h after the stress treatments (Figure 7a). The same observations as in the Pro35S:HsfA2 plants were obtained in other transgenic lines (Pro35S:HsfA2–3 and -12) (data not shown). However, at 8 h after stress treatments, no significant differences in visible leaf injury were observed between wild-type, KO-HsfA2 and Pro35S:HsfA2–6 plants (data not shown). No significant differences in PSII activity [variable fluorescence (Fv)/maximal fluorescence (Fm)] were detected between Pro35S:HsfA2–6, KO-HsfA2 and wild-type plants under normal conditions (Figure 7b). At 5 h after stress treatments, PSII activities of both wild-type and KO-HsfA2 plants decreased to approximately 2%, respectively, while that in the Pro35S:HsfA2–6 plants remained high.

image

Figure 7.  Evaluation of the environmental stress tolerance ofPro35S:HsfA2 and KO-HsfA2 plants. (a) Stress tolerance of wild-type, Pro35S:HsfA2 and KO-HsfA2 plants. Arabidopsis plants were grown under normal conditions for 2 weeks on MS medium, then transferred to soil culture under normal conditions (100 μE m−2 sec−1 at 20°C) for 1 week. Methylviologen (50 μm) in 0.1% Tween 20 (5 ml) was sprayed on wild-type, Pro35S:HsfA2 and KO-HsfA2 plants, which were then transferred to high-light and heat-shock (HL + HS) stress conditions for the times indicated. (b) Effects of stress on PSII activity in wild-type, Pro35S:HsfA2 and KO-HsfA2 Arabidopsis plants. The PSII activity (Fv/Fm) in the rosette leaf in Arabidopsis plants was determined at 25°C after dark adaptation for 30 min. Data are mean values ± SD of three individual experiments. Different letters indicate significant differences (P < 0.05).

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These results indicate that overexpression of HsfA2 in transgenic plants improves tolerance to combined environmental stresses, and that HsfA2 plays an important role as a key regulator in the induction of defence systems in response to these environmental stresses.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We isolated a large number of HL + HS stress-inducible genes, including HSPs and HsfA2 genes, by SSH (Table S1). Transcripts of the HsfA2 gene rapidly accumulated under HL + HS, HS, HL or CH stress conditions (Figure 1a). The strongest GUS activity was detected mainly in the rosette leaves of ProHsfA2:GUS plants under HL + HS stress conditions (Figure 3c and d), suggesting that HsfA2 functions in the gene-regulation system of leaves in response to stress conditions.

Recently, it has been reported from a genome-wide analysis of Arabidopsis (Vanderauwera et al., 2005) that HSPs and HsfA2 are H2O2-regulated genes. We also found that treatment with H2O2 increased the transcript levels of some HSPs and HsfA2 (Figures 1b and 5b). It is well known that a wide range of environmental stresses cause enhanced production of H2O2, which is a second messenger in plant cells (Dat et al., 1998; Levine et al., 1994; Prasad et al., 1994). Pnueli et al. (2003) reported that, in an Arabidopsis mutant that had a knockout of cytosolic APX (APX1), a key enzyme in the active oxygen species (AOS) scavenging system, the induction of many HSP genes and two HSF genes (including HsfA2) under HL stress was much higher compared with that in wild-type plants. Recently, it has been reported that transcript levels of HsfA2 and some HSPs increased along with an increase of H2O2 level during HS stress in Arabidopsis plants (Li et al., 2005). Based on the present data and on data reported previously, it seems likely that the increase in transcript level of HsfA2 is mediated by the increase in cellular H2O2 accumulation under several environmental stresses in Arabidopsis plants.

Kovtun et al. (2000) showed that H2O2 activated the Arabidopsis mitogen-activated protein kinase kinase kinase, ANP1, and the expression of a constitutively active ANP1 in Arabidopsis protoplasts led to the upregulation of promoter activity of Hsp18.1-CI, which was also identified as an HsfA2 target gene (Table 1; Figure 4). Recently, it has been reported that NDP kinase 2 (NDPK2) is associated with H2O2-mediated mitogen-activated protein kinase signalling in Arabidopsis plants (Moon et al., 2003). Furthermore, it has been reported that the expression of many types of transcription factor, including members of the WRKY, Zat, RAV, GRAS and Myb families, is enhanced by AOS (Desikan et al., 2001; Epple et al., 2003; Panchuk et al., 2002; Pnueli et al., 2003; Rizhsky et al., 2003, 2004b; Vandenabeele et al., 2003, 2004, Vanderauwera et al., 2005; Vranova et al., 2002). It is very interesting how such a kinase cascade and these transcription factors are associated with HsfA2 in response to environmental stresses in plants.

Recently, Li et al. (2005) have reported that HsfA2 modulated the expression of some HSPs and APX1 in Arabidopsis plants. To understand the more detailed molecular mechanisms of HsfA2 under environmental stress conditions, we identified the set of HsfA2 target genes using the transformants overexpressing HsfA2. Fifteen HL + HS stress-inducible genes were induced in the Pro35S:HsfA2 plants, even under normal conditions (Table 1; Figure 4). Furthermore, the transcripts of 37 genes were induced by DNA microarray analysis, and nine genes decreased in Pro35S:HsfA2–6 plants compared with wild-type plants under normal conditions (Tables S2 and S3). Thus the transcript levels of a total of 46 genes were upregulated in the Pro35S:HsfA2 plants. Recently, using the microarray analysis, the downregulated genes were identified in KO-HsfA2 plants under HS stress conditions (Schramm et al., 2006).

The expression levels of putative HsfA2 target genes were correlated with the level of HsfA2 transcripts (Figures 4 and 5). The putative promoter regions of all putative HsfA2 target genes also possessed perfect or imperfect HSE sequences (Table S4). The induction of expression of 26 genes was significantly reduced in the KO-HsfA2 Arabidopsis plants under HL + HS stress conditions compared with that of wild-type plants (Figure 6), indicating that these genes are directly regulated by the HsfA2 protein at an early stage under HL + HS stress conditions. These genes are many HSPs, cold-regulated protein (KIN2), APX2, GolS1GolS2, USP, and so on. It is well known that APX, GolS and HSPs play essential roles in the scavenging of AOS, the synthesis of osmolyte and the stabilization of damaged proteins, thereby assisting the cell's recovery from stress (Mittler, 2002; Schöffl et al., 1998; Shigeoka et al., 2002; Taji et al., 2002). On the other hand, we observed no significant difference in the transcript levels of 12 genes between wild-type and KO-HsfA2 plants under HL + HS stress conditions (data not shown). Because these genes also have HSEs in their promoter regions, other class A HSFs may also be involved in the expression of these genes under these stress conditions.

Among HsfA2 target genes, 11 genes, such as HSPs, APX2, GolS1 and GolS2, were the same as HsfA1a/1b target genes (Table 1; Busch et al., 2005; Panchuk et al., 2002; Panikulangara et al., 2004; Wunderlich et al., 2003). The HsfA2 expression was highly induced compared with other class A HSFs at the early stage of HL + HS stress conditions, while the transcript levels of HsfA1a and HsfA1b were not changed under HL + HS stress conditions (Figure 2). These results suggest that the HsfA2 protein substantially acts for the rapid induction of expression of these target genes under these stress conditions. In contrast, constitutive expression of the dominant-negative construct for HSF21 (HsfA4a) prevented the accumulation of transcripts encoding the APX1 and the zinc-finger protein Zat12 in response to light stress, while the induction of Hsp70 (At2g32120) expression as an HsfA2 target gene was not affected under light-stress conditions (Davletova et al., 2005). These findings indicate that the target genes of HsfA4a are clearly different from those of HsfA2.

The KO-HsfA2 plants did not show high sensitivity to HL, HS or CH stresses (data not shown); and the KO-HsfA1a/1b plants did not exhibit a strong loss of thermotolerance (Lohmann et al., 2004). The transcript level of HsfA2 was remarkably induced in the KO-HsfA1a/1b plants to the same extent as in the wild-type plants under HS stress conditions (Busch et al., 2005). A slow induction of target genes was observed in KO-HsfA1a/1b or KO-HsfA2 under stress conditions (Figure 6; Lohmann et al., 2004). These results suggest that other class A HSFs may compensate for the role of HsfA1a/1b or HsfA2.

It has been reported that the HsfA2 overexpressing transgenic Arabidopsis plants showed increased tolerance to HS and oxidative stresses compared with wild-type plants (Li et al., 2005). However, Pro35S:HsfA2 plants did not show increased tolerance to HL, HS or CH stress conditions (data not shown). The discrepancy between the previous data and our present data may be caused by a difference in experimental conditions.

The Pro35S:HsfA2 plants showed increased tolerance to combined stress conditions, HL + HS plus MV treatments, up to 5 h (Figure 7). Modification of stress tolerance in Pro35S:HsfA2 plants can be accounted for by the upregulation of several stress-inducible genes before the stress treatments (Table 1). In fact, the overexpression of these genes leads to resistance to various types of stress being enhanced (Prändl et al., 1998; Sun et al., 2001; Taji et al., 2002; Webb and Allen, 1996). The PSII activity in KO-HsfA2 plants was lower than that in wild-type plants at 3 h after HL + HS plus MV treatments (Figure 7). However, no significant differences in visible leaf injury were observed between wild-type, KO-HsfA2 and Pro35S:HsfA2–6 plants at 8 h after stress treatments (data not shown). It is noteworthy that the transcript levels of HsfA2 and several HsfA2 target genes are transiently increased by HL + HS stress (Figure 6). These findings, including the present data, strongly indicate that HsfA2 is a key regulator in rapid induction of the defence system through H2O2 at early stage in response to these environmental stresses.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and stress treatments

Arabidopsis thaliana ecotype Columbia plants were grown at 16 h light, 25°C/8 h dark, 22°C on Murashige and Skoog (MS) medium under a light intensity of 100 μE m−2 sec−1. Two-week-old seedlings were subjected to various stress conditions. The knockout Arabidopsis lines containing a T-DNA insert in the HsfA2 gene (KO-HsfA2; obtained through the SIGnAL project; http://signal.salk.edu/tabout.html) were outcrossed and selfed to check for segregation and to obtain pure homozygous lines, as described (Sussman et al., 2000). Analysis of the HsfA2 knockout and segregation were performed with PCR and a genomic Southern blot. The combination of HL + HS stress treatments was accomplished by exposure to 800 μE m−2 sec−1, 40°C in a growth cabinet (Biotrom NC350; NK System, Osaka, Japan). The HL stress was accomplished by 800 μE m−2 sec−1, 25°C in a growth cabinet. The HS stress was carried out by raising the temperature to 40°C under dark conditions. The CH stress was carried out by lowing the temperature to 4°C under conditions of 100 μE m−2 sec−1. Plants were collected and frozen in liquid nitrogen and stored at −80°C for further preparation.

Subtractive hybridization

Total RNA was extracted from the aerial parts of seedlings, which were treated with HL + HS stress (800 μE m−2 sec−1, 40°C for 1 h) and normal conditions (100 μE m−2 sec−1, 25°C). Poly(A)+ RNA (2 μg) was purified using PolyATract mRNA Isolation Systems (Promega, Madison, WI, USA) and used for subtractive hybridization according to the Clontech PCR-Select Subtractive Hybridization kit manual (Clontech, Palo Alto, CA, USA). After PCR amplification, the PCR products were inserted into the pSTBlue-1 AccepTor Vector (Novagen, Madison, WI, USA). DNA sequences were determined using the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences of DNA and predicted amino acids were searched against the DNA and protein databases using the blast programs available at the NCBI website (http://www.ncbi.nlm.nih.gov).

Reverse Northern analysis

Reverse Northern analysis was performed according to the method described by Zhang et al. (1996). cDNA inserts were individually PCR-amplified and transferred to Hybond N+ membrane (Amersham Biosciences, Uppsala, Sweden). The λDNA was used as a negative control. Each total cDNA probe was synthesized from 5 μg total RNA using PowerScript Reverse Transcriptase (Clontech). The membrane was hybridized with [α-32P]-dCTP labelled cDNA probes from control and stressed plants at 65°C for 16 h. The hybridized membrane was washed in 2 × standard saline citrate (SSC), 0.2 × SSC, 0.1% SDS at 68°C, and autoradiography was carried out with Mac BAS 1000 (Fuji Photofilm, Tokyo, Japan).

RNA gel-blot analysis

Total RNA (20 μg) was fractionated in a 1.2% agarose gel containing 2.2 m formaldehyde and transferred to a Hybond N+ membrane (Amersham). Prehybridization took place at 60°C for 3 h in the buffer containing 6 × SSC, 1 × Denhard's solution, 1% SDS and 100 μg ml−1 denatured salmon sperm DNA. The membrane was hybridized with [α-32P]-dCTP labelled probes at 60°C for 16 h. The hybridized membrane was washed in 2 × SSC, 0.2 × SSC, 0.1% SDS at 60°C, and autoradiography was carried out with Mac BAS 1000.

Generation of transgenic plants

The pBI/Pro.35S:HsfA2 plasmid was constructed as follows. The coding region of HsfA2 cDNA was amplified by reverse transcriptase-polymerase chain reaction using primers 5′-CTCTGAGCTTATGGATTTGAG-3′ and 5′-GACCGCAACAAGTAGATGTG-3′. The amplified fragment was integrated into the XbaI/KpnI site between the CaMV35S and the nopalin synthetase (NOS) terminator sequence of the plant binary vector pBI121 (Clontech). To examine the tissue-specific expression of HsfA2, the pBI/Pro.HsfA2:GUS construct was obtained by amplifying 2 kbp of the HsfA2 promoter region using primers 5′-GTCGACCTTTGCCAATTCCT-3′ (SalI site in italics) and 5′-GGATCCTTTCGTTGTTTATC-3′ (BamHI site in italics) and cloning the amplified DNA into the pBI101.3 vector (Clontech). DNA sequences were confirmed using the ABI Prism 3100 Genetic Analyzer. Arabidopsis thaliana ecotype Columbia plants were transformed using the Agrobacterium harbouring the pBI/Pro.35S:HsfA2 and pBI/Pro.HsfA2:GUS constructs. T3 seeds were used for subsequent experiments.

Histochemical analysis

ProHsfA2:GUS transgenic Arabidopsis plants were grown at 16 h light, 25°C/8 h dark, 22°C on MS medium under a light intensity of 100 μE m−2 sec−1. Two-week-old ProHsfA2:GUS transgenic plants were transferred to 800 μE m−2 sec−1, 40°C for 3 h. Histochemical localization of GUS activity in transgenic plants was performed by incubating whole seedlings in 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) buffer [100 mm sodium phosphate buffer pH 7.0, 0.5 mm K3Fe(CN)6, 0.5 mm K4Fe(CN)6, 0.3% Triton X-100, 20% methanol, 1.9 mm X-gluc] at 37°C for 1–3 h. Seedlings were washed with 70% ethanol until they were cleared of chlorophyll, then examined using VB-G25 light microscopy (Keyence, Osaka, Japan).

Microarray analysis

The wild-type and Pro35S:HsfA2–6 plants were grown under normal conditions, as described above. Total RNA was isolated using Sepasol-RNA I (Nakarai tesque, Kyoto, Japan) from 10 to 20 wild-type and Pro35S:HsfA2–6 plants; mRNA was prepared using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The total cDNA probe was synthesized and labelled using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) following the manufacturer's instructions. Two differentially labelled samples were injected into a microarray hybridization chamber loaded with a 22 K Arabidopsis2 60-mer oligo microarray (Agilent Technologies), hybridized for 17 h at 60°C, and washed according to the manufacturer's instructions. Hybridization microarray slides were scanned with a ScanArray 4000XL (GSI Lumonics, Oxnard, CA, USA). The images generated were analysed using QuantArray (GSI Lumonics), applying standard normalization procedures.

Quantitative PCR analysis

Following HL + HS stress treatments, total RNA was isolated as described above. To eliminate any DNA contamination, 50 μg total RNA was treated with DNase I (Takara, Kyoto, Japan). Total RNA was purified with an RNeasy Plant Mini Kit (Qiagen) and converted into cDNA using the ReverTra Ace (Toyobo, Osaka, Japan) with the oligo (dT)20 primer. Primer pairs for quantitative PCR were designed using primer express software (Applied Biosystems); primer sequences are shown in Table S5. Gene-specific primers were chosen such that the resulting PCR product had an approximately equal size of 100 bp. Quantitative PCR was performed with an Applied Biosystems 7300 Real Time PCR System, using the SYBR Premix Ex Taq (Takara). Actin2 mRNA, set to 100%, was used as an internal standard in all experiments. Quantitative PCR experiments were repeated at least three times for cDNA prepared for three batches of plants.

Stress tolerance of transgenic plants

Arabidopsis plants were grown under normal conditions for 2 weeks on MS medium, as described above, then transferred to soil culture under normal conditions (100 μE m−2 sec−1, 20°C) for 1 week. Three-week-old plants were exposed to HL + HS stresses (45°C, 1600 μE m−2 sec−1) plus the treatment of 50 μm MV.

Chlorophyll fluorescence measurements

Chlorophyll fluorescence was measured according to the method described by Miyagawa et al. (2000). Chlorophyll fluorescence in the third leaf in the tobacco plants was measured at 25°C with a Mini PAM Chl Fluorometer (Waltz, Efeltrich, Germany).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Drs Fang-Sik Che and Akira Isogai for use of the DNA sequencing analyser. We also thank Drs Miho Takemura and Akiho Yokota for their kind help in using the microarray system. This work was supported by CREST, JST (2005–2010) and by the ‘Academic Frontier’ Project for Private Universities: matching fund subsidy from MEXT, 2004–2008.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 Upregulated genes under a combination of high-light and high-temperature stress conditions in Arabidopsis leaves

Table S2 Upregulated genes in Pro35S:HsfA2–6 plants compared with wild-type plants under normal conditions

Table S3 Downregulated genes in Pro35S:HsfA2–6 plants compared with wild-type plants under normal conditions

Table S4 Existence of the heat-shock element in HsfA2 target genes

Table S5 Primer used for quantitative PCR analysis

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