JXB Advance Access published online on September 20, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm184
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth
1Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan
2Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan
* To whom correspondence should be addressed. E-mail tnish9{at}kenroku.kanazawa-u.ac.jp
Received 23 May 2007; Revised 3 July 2007 Accepted 16 July 2007
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Abstract |
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Heat shock transcription factors (Hsfs) are the central regulators of the heat shock (HS) stress response in all eukaryotic organisms. HsfA2 is one of the Arabidopsis class A Hsfs, and the induction of HsfA2 expression in response to HS stress is highest among all 21 Arabidopsis Hsfs. In this study, it is reported that basal and acquired thermotolerance was significantly enhanced in high-level HsfA2-overexpressed transgenic lines (El2
::HsfA2) in comparison with wild-type plants. By contrast, the dominant negative mutants of HsfA2 (El2::HsfA2
C264) plants displayed reduced thermotolerance. These results indicate that the HsfA2 gene plays a role in the HS stress response. Microarray analysis of the El2::HsfA2 plants identified putative target genes, which included HS stress-inducible genes and other stress-responsive genes. Salt and osmotic stress induced HsfA2 gene expression. In fact, the El2::HsfA2 plants showed enhanced tolerance to these stresses, suggesting that HsfA2 was involved in multiple stress tolerance. El2::HsfA2 plants showed accelerated callus growth from root explants compared with the wild type, unlike the El2::HsfA2C264 plants whose growth was delayed. These observations suggest that HsfA2 plays, in addition to its role in stress tolerance, an important role in cell proliferation. Key words: Biomass, callus, heat shock response, Hsf, microarray, osmotic stress, salt stress
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Heat shock transcription factors (Hsfs) are the central regulators of the heat shock (HS) stress response in all eukaryotic organisms (Baniwal et al., 2004). Hsfs have a common structure with an N-terminal DNA binding domain (DBD), a hydrophobic coiled-coil region (HR-A/B) essential for oligomerization, one or two nuclear localization signals, and a C-terminal activation domain (CTAD). Hsfs specifically bind to cis-elements termed heat shock elements (HSEs), which consist of alternating units of the sequence (5'-nGAAn-3'), and subsequently activate the transcription of HS-inducible genes like the genes encoding heat shock proteins (Hsps) (Wu, 1995; Morimoto, 1998; Schöffl et al., 1998; Nover et al., 2001; Baniwal et al., 2004). Hsps function as molecular chaperones that play a crucial role in many cellular processes (Hartl and Hayer-Hartl, 2002; Wang et al., 2004).
Only a few Hsf genes have been identified in animals (Morimoto, 1998; Nakai, 1999), whereas genomes of Arabidopsis and rice contain 21 and 23 different Hsf genes, respectively (Nover et al., 2001; Kotak et al., 2004). In tomato and soybean, multiple Hsf genes have been identified from expressed sequence tag databases (Nover et al., 2001; Kotak et al., 2004). These plant Hsfs are classified into three major classes (A, B, and C) based on the length of the flexible linker region between the DBDs and the HR-A/B regions and the number of amino acid residues inserted into the HR-A/B regions (Nover et al., 2001). All class A Hsfs are assumed to be transcriptional activators because of the presence of AHA motifs in their CTADs. AHA motifs are activator peptide motifs containing characteristic patterns of aromatic, large hydrophobic, and acidic amino acid residues and have been found in many other transcriptional activators of yeast and mammals (Döring et al., 2000). In fact, it was reported that some Arabidopsis and tomato class A Hsfs function as transcriptional activators in tobacco protoplasts and yeast (Treuter et al., 1993; Döring et al., 2000; Kotak et al., 2004). By contrast, class B Hsfs lacking AHA motifs have no capacity to activate transcription in tobacco protoplasts and yeast (Czarnecka-Verner et al., 2000; Kotak et al., 2004). However, there is evidence that HsfB1 of tomato and Arabidopsis act as a transcriptional coactivator and a transcriptional repressor, respectively (Czarnecka-Verner et al., 2000, 2004; Bharti et al., 2004). These facts imply that class B Hsfs are also involved in transcriptional regulation. On the other hand, the functions of class C Hsfs are largely unknown. Thus, the multiplicity of plant Hsfs suggests a functional diversity and complexity in plant Hsfs.
Overexpression of HsfA1a or HsfA1b in Arabidopsis constitutively caused weak expression of Hsps under normal conditions and resulted in enhanced basal thermotolerance (Lee et al., 1995; Prändl et al., 1998). By contrast, hsfA1a hsfA1b double mutants exhibit a weak loss of thermotolerance (Lohmann et al., 2004). Among these class A Hsfs, expression of HsfA2 is most strongly induced by HS stress (Busch et al., 2005). Recently, several groups have reported the in vivo function of HsfA2 by analysing HsfA2 overexpressing transgenic plants and T-DNA insertion mutants in HsfA2. However, there are some discrepancies between the data presented in these reports. Li et al. (2005) noted that hsfA2 mutants displayed reduced basal and acquired thermotolerance as well as oxidative stress tolerance, while transgenic plants overexpressing HsfA2 displayed increased tolerance to these stresses. Nishizawa et al. (2006) and Schramm et al. (2006) reported no obvious phenotype for thermotolerance when the same mutants were used in their experimental conditions. In addition, there was no obvious difference in the thermotolerance between the HsfA2 overexpressing plants and wild-type plants, except for an extremely severe stress condition: a combination of high light, HS, and methylviologen (Nishizawa et al., 2006).
In this study, El2::HsfA2 transgenic Arabidopsis plants, whose expression level is higher than that of previous reports, were generated. These transgenic plants exhibited a dwarf phenotype and remarkable basal and acquired thermotolerance, although no visible phenotype was observed in previously reported HsfA2-overexpressed plants driven by a single 35S promoter (Li et al., 2005; Nishizawa et al., 2006). The El2::HsfA2C264 plants carrying a dominant negative form of HsfA2 displayed reduced basal and acquired thermotolerance and reduced expression levels of the putative HsfA2 target genes even under HS stress. These results suggest that the HsfA2 gene plays important roles in the HS stress response. In addition, the HsfA2 gene and other class A genes may be functionally redundant. It was found that the El2::HsfA2 plants exhibit tolerance to salt and osmotic stress, suggesting that HsfA2 was involved in these stress responses. Interestingly, El2::HsfA2 plants showed accelerated callus growth from roots compared with the wild type.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant materials and growth conditions
Arabidopsis thaliana (ecotype; Col-0) was used in this study. Wild-type and transgenic R2 seeds were surface-sterilized with 1% (v/v) PPM (Plant Cell Technology, Inc.). Sterile seeds were sown on Murashige and Skoog (MS) medium that contained 2% (w/v) sucrose and 0.2% (w/v) gelrite (San-Ei Gen F.F.I., Inc.) in plastic Petri dishes. Then they were stratified for 2 d at 4 °C in the dark. Plants were grown at 22 °C under long-day conditions (16 h light/8 h dark cycles or continuous light) in a growth chamber.
Generation of transgenic plants
To generate El2::HsfA2 plants, the coding region of HsfA2 was amplified by PCR using the following primers: forward primer, 5'-TCTAGAATGGAAGAACTGAAAGTGG-3'; reverse primer, 5'-GAGCTCTTAAGGTTCCGAACCAAGA-3' (underlined sections are XbaI and SacI sites, respectively). To generate El2::HsfA2C264, the cDNA encoding the C-terminal-deleted HsfA2 1–264 protein was amplified by PCR using the following primers: forward primer, 5'-TCTAGAATGGAAGAACTGAAAGTGG-3'; reverse primer, 5'-GAGCTCTTACTCTTGATCATGTAACAAA-3' (underlined sections are XbaI and SacI sites, respectively). The PCR products were cloned into the SmaI site of pUC19 and checked by sequencing. After digestion of the resultant plasmid with XbaI/SacI, the fragments were purified and cloned into the XbaI/SacI site of the binary vector pBE2113-GUS (Mitsuhara et al., 1996). The resultant constructs (pBE2113-HsfA2 and pBE2113-HsfA2C264) were introduced into Agrobacterium tumefaciens strain GV2260. Arabidopsis were transformed with these constructs by the floral dip method (Clough and Bent, 1998). Transgenic R1 plants were selected on MS plates that contained 50 µg ml–1 of kanamycin.
Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen), and on-column DNA digestion was performed using the RNase-free DNase set (Qiagen). In a total volume of 20 µl, cDNAs were synthesized from 1 µg of total RNA using SuperScript III reverse transcriptase (Invitrogen) with an oligo(dT)16 primer, and then 0.5 µl of the cDNA was used in the subsequent PCR. All PCR were performed in a total volume of 10 µl for 22–28 cycles under the following conditions: denaturation, 94 °C, 30 s; annealing, 60 °C, 30 s; extension, 72 °C, 30 s. The following gene-specific primers were used: HsfA2: 5'-TGGTGTGCTTGTAGCTGAGG-3' and 5'-CATAACCGCAAACTGCTGAA-3'; ELIP1: 5'-CCTCGGTACAACAGCGATCT-3' and 5'-ACGAGTGTCCCACCTTTGAC-3'; GolS1: 5'-AAACCGCTGATGCTATGTCC-3' and 5'-TCACGTAATCACCGTTTCCA-3'; HsfB1: 5'-TCGTGTGGAAAACAGCAGAG-3' and 5'-ATTTACGCCGTCGTATGTCC-3'; HsfB2a: 5'-AACGAAGACGACTGGGAATG-3' and 5'-CGGGAGAATAACTCCGATCA-3'; Hsp90-1: 5'-GCTGCTAGGATTCACAGGATG-3' and 5'-TCCTCCATCTTGCTCTCTTCA-3'; HSP101: 5'-TAACGGGCCAAAGAGAAGTG-3' and 5'-CAAACGTTGGAGGTCAAGACT-3'; APX2: 5'-AAGTTGAGCCACCTCCTGAA-3' and 5'-GTGTGTCCACCAGACAATGC-3'; MBF1c: 5'-AGAGGTGAGGTTGATGATAC-3' and 5'-GCACAGCCTGATTAGGA-3'; Actin2/8: 5'-GGTAACATTGTGCTCAGTGGTGG-3' and 5'-AACGACCTTAATCTTCATGCTGC-3'; UBQ5: 5'-CTTGAAGACGGCCGTACCCTC-3' and 5'-CGCTGAACCTTTCAAGATCCATCG-3'. All kits were used according to the manufacturer's protocol.
Real-time PCR
Real-time PCR was performed using the LightCycler Quick System 350S (Roche Diagnostics K.K.) with SYBR Premix Ex Taq (Takara Bio, Inc.). Total RNA isolation, cDNA synthesis, and primer sequences were as described above. Each PCR reaction contained 1x SYBR Premix Ex Taq, 0.2 µM of each primer, and 2 µl of a 1:5 dilution of the cDNA in a final volume of 20 µl. The following PCR programme was used: initial denaturation, 95 °C, 10 s; PCR, 40 cycles of 95 °C, 5 s, 60 °C, 20 s with a temperature transition rate of 20 °C s–1. In melting curve analysis, PCR reactions were denatured at 95 °C, reannealed at 65 °C, then a monitored release of intercalator from PCR products or primer dimmers by an increase to 95 °C with a temperature transition rate of 0.1 °C s–1. To create a standard curve, homologous standards for each gene were used as external standards in all experiments. cDNA quantities were calculated by the second derivative maximum methods of LightCycler Software Ver.3.5 (Roche Diagnostics), and all quantifications were normalized using Actin2/8 mRNA as an internal control.
Stress assays
For the basal thermotolerance assays and acquired thermotolerance assays, wild-type and transgenic plants grown on MS medium in plastic Petri dishes were used and incubated in the climate chambers. For the basal thermotolerance assays, 5-d-old plants were exposed to HS stress at 45 °C for 90 min, and then were grown at 22 °C. After HS treatment, the surviving plants were counted daily. Alternatively, 10-d-old plants were exposed to HS stress at 45 °C for 60 min, were grown at 22 °C for 5 d, and were then photographed. For acquired thermotolerance assays, 5-d-old plants were exposed to a pre-conditioning heat treatment at 37 °C for 1 h and incubated at 22 °C for 3 h, and then exposed to HS stress at 49 °C for 90 min. After HS treatment, these plants were grown at 22 °C. The number of surviving plants was scored daily after HS treatment. For each experiment, more than 50 plants were used and values represent the means of three experiments. Differences in thermotolerance were confirmed as being statistically significant using Student's t test.
For the analysis of tolerance to salt and osmotic stresses, seeds of wild-type plants and El2::HsfA2 plants were surface-sterilized and sown on MS medium containing different concentrations of NaCl (0–150 mM) or mannitol (0–400 mM). After stratification for 2 d at 4 °C in the dark, plates were maintained in a climate chamber (22 °C, 16 h light/8 h dark cycles), and then their root lengths were scored after 7 d. Sixteen plants were used for each experiment. The relative root length of NaCl- or mannitol-treated plants to untreated plants were compared between wild-type and El2::HsfA2 plants. Differences in salt and osmotic stress tolerance were confirmed as being statistically significant using the Mann–Whitney U-test.
For expression analysis, 10-d-old wild-type and transgenic plants grown on MS medium were used in all stress treatments. For HS stress treatment, plants were exposed to 37 °C for 1 h in a climate chamber. For NaCl treatment, plants were transferred onto filter paper saturated with 250 mM NaCl in MS liquid medium for 6, 12, and 24 h. For mannitol treatment, plants were transferred onto filter paper saturated with 400 mM mannitol in MS liquid medium for 6, 12, and 24 h. Whole plants were collected, frozen with liquid nitrogen, and stored at –80 °C.
Microarray analysis
Total RNAs were isolated from aerial parts of 2-week-old wild-type plants and two independent El2::HsfA2 lines (#2 and #24) grown on MS agar plates using the RNeasy Plant Mini Kit (Qiagen). The quality of RNA samples was assessed with the RNA 6000 Nano LabChip Kit (Bioanalyser 2100; Agilent Technologies, Inc.), then subjected to microarray experiments using the Agilent Arabidopsis 2 22 k Oligo Microarray (Agilent Technologies, Inc.). Microarray analyses were performed according to the Agilent 60-mer Oligo Microarray Processing Protocol (Agilent Technologies, Inc.). Total RNAs (400 ng) of wild-type and El2::HsfA2 lines were used for the preparation of Cy3- or Cy5-labelled cRNA using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Two different fluorescent-labelled cRNAs were combined and purified with the use of the RNeasy RNA purification Kit (Qiagen, Inc.). Hybridized and washed arrays were scanned with maximum laser intensity in both the Cy3 and Cy5 channels using an Agilent microarray scanner (G2565BA; Agilent Technologies). Images were analysed with Feature Extraction Software (version 7.5; Agilent Technologies). Two independent experiments were carried out using two independent transgenic lines (#2 and #24) to achieve good reproducibility of microarray analyses. In addition, colour swap in each experiment was performed. Up-regulated genes were determined by a greater than 6-fold change in their expression ratio (El2::HsfA2 plants versus wild type) in all four arrays. All of these changes in gene expression were statistically significant at P <0.01.
Callus formation from root explants of Arabidopsis
Arabidopsis seedlings were grown for 7 d on MS agar medium (Che et al., 2002). Roots were cut into 5 mm segments which were transferred to callus-inducing medium (CIM) (Che et al., 2002). Then, root explants were incubated on CIM at 22 °C under long-day conditions (16 h light/8 h dark cycles or continuous light) in a growth chamber.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Generation of highly HsfA2-overexpressed transgenic Arabidopsis plants
To investigate the in vivo function of HsfA2, transgenic plants were generated that overexpressed the HsfA2 gene under the control of the two tandem repeats of an enhancer-like sequence of cauliflower mosaic virus 35S promoter, 35S promoter, and the tobacco mosaic virus
sequence to enhance the efficiency of translation (Mitsuhara et al., 1996). Twenty-eight T1 transgenic lines could be classified into three classes: nine lines were severe dwarf, 10 lines were weak dwarf, while nine lines were visibly normal (data not shown). Three severely dwarfed lines (El2::HsfA2#2, #24, and #25) and two weakly dwarfed lines (El2::HsfA2#13 and #19) among these transgenic lines were selected. Three transgenic plants (#2, #24, and #25) showed a markedly higher expression (>430-fold) of the HsfA2 gene compared with wild type, while two transgenic lines (#13 and #19) showed higher expression (60-fold and 140-fold, respectively) than wild type (Fig. 1A). The severity of the dwarf phenotype among the El2::HsfA2 lines was dependent on the expression level of the HsfA2 gene (Fig. 1B). By contrast to the El2::HsfA2 plants in the present study, 35S::HsfA2 plants used in other laboratories were reported to exhibit no visible phenotype in unstressed conditions (Li et al., 2005; Nishizawa et al., 2006). It was likely that expression of the HsfA2 gene in the El2::HsfA2 plants in the present study was higher than in the 35S::HsfA2 plants of previous reports. In fact, the transgenic plants of the present study, which were regulated by the 35S promoter alone, showed no obvious dwarfism (data not shown). The tobacco mosaic virus sequence may be effective in the translation of the HsfA2 protein.
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The primary roots in these lines were slightly shorter than those in the wild type (Fig. 1C), whereas the number of their lateral root branches increased without any apparent morphological change (data not shown). In addition, bolting and flowering of El2
::HsfA2 plants were delayed by several days to some weeks as compared with wild-type plants (data not shown). Although the organ identities and numbers of El2::HsfA2 flowers were normal, flower morphology was altered (data not shown). These results indicate that high-level overexpression of HsfA2 affected both vegetative and reproductive growth.
Generation of transgenic Arabidopsis plants overexpressing a dominant negative form of HsfA2
The hsfA2 mutant, in which a T-DNA was inserted 745 bp downstream of the translational start site of the HsfA2 gene, was obtained from the Arabidopsis Biological Resource Center. No significant differences were observed between the wild-type plants and the hsfA2 mutant plants even under HS stress conditions in the present experimental conditions (data not shown). Similar results were also reported by two independent research groups (Nishizawa et al., 2006; Schramm et al., 2006). For further analysis of the HsfA2 function in thermotolerance, El2::HsfA2C264 plants were generated, which express a dominant negative form of HsfA2 containing the DBD, oligomerization domain, and nuclear localization signal, but not the transcriptional activation domain and nuclear export signal (Fig. 2A). There were no significant differences in appearance between the wild-type and El2::HsfA2C264 plants under normal growth conditions (data not shown).
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El2
::HsfA2 plants exhibit enhanced basal and acquired thermotoleranceThe basal and acquired thermotolerance of El2
::HsfA2 and El2::HsfA2C264 plants were then compared with that of wild-type plants. Without pre-conditioning heat treatment, most of the wild-type plants were killed at 45 °C after 5 d of HS treatment (Fig. 2B), while all El2::HsfA2#25 plants could survive after 5 d of the same treatment (Fig. 2B). Similar results were also obtained in El2::HsfA2#2 and #24 plants (data not shown). After pre-conditioning heat treatment, all El2::HsfA2#25 plants could survive at 49 °C, although most of the wild-type plants could not (Fig. 2C). Thus, a high level of overexpression of HsfA2 could confer increased basal and acquired thermotolerance to Arabidopsis plants. On the other hand, a significant difference in thermotolerance between El2::HsfA2#13 and wild-type plants was observed only after 5 d of HS treatment at 45 °C without pre-conditioning heat treatment (Fig. 2B, C). These results indicate that the extent of thermotolerance was dependent on the expression level of the HsfA2 gene among El2::HsfA2 lines (Fig. 1A). Figure 2D shows that El2::HsfA2 plants exhibited a dwarf phenotype but enhanced thermotolerance in comparison to wild-type plants. Nishizawa et al. (2006) observed no obvious difference in the visible phenotypes and thermotolerance between the wild-type and HsfA2-overexpressing plants, but increased tolerance to combined stress of high light, HS, and methylviologen. Therefore, it is likely that the phenotypic differences between the HsfA2-overexpressed lines in the present study and those reported in the literature were dependent on the expression level of HsfA2.
Concerning basal thermotolerance, the survival rates of the El2::HsfA2C264#2 (x90) plants were significantly lower than those of wild-type plants at 45 °C after 4 d and 5 d of HS treatment (Fig. 2B). As for acquired thermotolerance, survival rates between the El2::HsfA2C264#2 and wild-type plants were also significantly different after 4 d and 5 d of HS treatment at 49 °C (Fig. 2C). Both basal and acquired thermotolerance were further reduced in the El2::HsfA2C264#14 (x150) line (Fig. 2B, C). The hypersensitivity to HS stress in transgenic lines was also dependent on the expression levels of a dominant negative form of HsfA2. Taken together, the HsfA2 gene plays important roles in the HS stress response, although this gene and other class A genes may be functionally redundant.
Expression profiling of El2::HsfA2 plants
Microarray analysis was performed using Agilent Arabidopsis 2 (22k) to obtain the expression profiles of putative HsfA2-regulated genes. This analysis was carried out using two independent wild-type plants, and two independent transgenic lines (El2::HsfA2#2 and El2::HsfA2#24) under the unstressed condition. Dye swap was performed to eliminate dye bias associated with unequal incorporation of Cy3 and Cy5 into cRNA. Then, putative HsfA2-regulated genes were designated as those with a greater than 6-fold induction in all four arrays. In El2::HsfA2 plants, 56 genes were up-regulated >6-fold compared with wild-type plants (Table 1). As seen in Table 1, 15 (27%) out of 56 up-regulated genes were annotated for Hsps. Six Hsp genes were up-regulated >100-fold. HSEs were commonly observed in the 5' upstream region of all 15 Hsps genes, suggesting that these genes were directly regulated by HsfA2. In addition, the microarray database (TAIR: http://www.arabidopsis.org/servlets/TairObject?type=expression_set&id=1007967124) suggests that 14 Arabidopsis Hsp genes were induced in response to HS stress within 6 h. It was reported that Hsp101 and Hsa32 genes were in fact involved in thermotolerance (Hong and Veirling, 2000; Hong et al., 2003; Charng et al., 2007).
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Table 1 also contains many stress-responsive genes such as APX2 and GolS1. The APX2 gene encodes cytosolic ascorbate peroxidase which scavenges reactive oxygen species (Mittler et al., 2004). Expression of the APX2 gene is induced by HS stress, high light, wounding, and a stress-related phytohormone, abscisic acid (Chang et al., 2004). The GolS1 gene encodes galactinol synthase, a rate-limiting enzyme of raffinose oligosaccharide (Taji et al., 2002). Multiple stresses such as HS stress, dehydration, and salt stress induced the expression of the GolS1 gene, resulting in the accumulation of raffinose oligosaccharide (Taji et al., 2002; Panikulangara et al., 2004). It is thought that raffinose oligosaccharide plays an important role in these stress responses as a compatible solute. In addition, the IPS2 gene product also participates in the same metabolic pathway (Busch et al., 2005). Therefore, up-regulation of these genes by HsfA2 might affect not only thermotolerance but also other stress responses in plants.
Interestingly, two class B Hsfs genes, HsfB1 and HsfB2a, were also up-regulated in El2::HsfA2 plants. Although class B Hsf proteins did not have AHA, tomato HsfB1 reportedly functioned as a transcriptional coactivator. Therefore, it is possible that HsfA2 and these class B Hsfs coordinately controlled their target genes. A multiprotein bridging factor 1c (MBF1c) gene, also indicated in Table 1, acted as a transcriptional coactivator. Expression of the MBF1c gene was induced by HS stress, salt stress, osmotic stress, and dehydration (Tsuda and Yamazaki, 2004). Since overexpression of the MBF1c gene in fact led to thermotolerance and osmotic tolerance in Arabidopsis (Suzuki et al., 2005), up-regulation of the MBF1c gene in El2::HsfA2 plants may play some role(s) in these stress responses.
As seen in Table 1, the target sequence (HSE, GAAnnTTC) of HsfA2 was frequently observed in the 5' upstream region (1 kb from 5' ATG) of 45 up-regulated genes (80%). Most of them contained multiple HSE elements (Table 1). By contrast, Busch et al. (2005) reported that HSE was found in the 5' upstream region (1 kb from 5' ATG) of 7611 genes (33%) in a total of 22 810 genes within the Arabidopsis genome. The possibility that most of the genes identified by the present microarray analysis might be directly regulated by HsfA2 is also supported by these data.
Nishizawa et al. (2006) reported a microarray analysis using the same Arabidopsis 2 array and found that in 35S::HsfA2 transgenic plants, 37 genes were up-regulated compared with wild-type plants under unstressed conditions. Of these 37 genes, 13 genes, including many Hsp genes, were also found in the 56 genes identified (Table 1). By contrast, putative transcriptional coactivator genes, HsfB1, HsfB2a, and MBF1c, were not within the group of 37 up-regulated genes of 35S::HsfA2 transgenic plants (Nishizawa et al., 2006). However, northern blot analysis showed that some up-regulated genes such as GolS1 in El2::HsfA2 plants were also up-regulated in the 35S::HsfA2 plants, as reported by Nishizawa et al. (2006). Therefore, high-level overexpression of the HsfA2 gene might have significantly affected the gene expression profile, and resulted in a difference in growth defects and thermotolerance between El2::HsfA2 and 35S::HsfA2 plants.
Most up-regulated genes in El2::HsfA2 plants are induced by HS stress
To verify the microarray results, the expression levels of eight up-regulated genes were examined in wild-type, El2::HsfA2, and El2::HsfA2C264 plants with or without HS treatment by semi-quantitative RT-PCR (Fig. 3A). In unstressed plants, the expression levels of these genes were apparently higher in El2::HsfA2 plants than those in the wild-type plants. Figure 3A also showed that all these genes exhibited a heat-inducible expression pattern in wild-type plants. It is likely that constitutive expression of these HS-responsive genes contributes to the enhanced thermotolerance observed in the El2::HsfA2 plants. Expression of these genes in El2::HsfA2 plants was also enhanced by HS treatment, probably through increased expression of the intrinsic HsfA2 gene and others in the HsfA family of genes by HS (Fig. 3A). The expression levels of several genes were compared between wild-type plants and four El2::HsfA2 lines by real time RT-PCR analysis. As expected, the expression levels of MBF1c, ELIP, and Hsp101 were dependent on that of HsfA2 (Fig. 3B).
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It was also found that the expression levels of all the genes tested were reduced in the El2
::HsfA2C264 plants compared with the wild-type plants under HS treatment (Fig. 3A). The overexpressed dominant negative form of HsfA2 may interact with other transcriptional activators containing native HsfA2 and consequently not allow any proper activation since it makes a functional binding but non-functional transcriptional activator. Therefore, the function of HsfA2 might be redundant with other transcriptional activators, probably class A Hsf proteins.
El2::HsfA2 plants exhibit enhanced salt and osmotic stress tolerance
Previous studies have demonstrated a link between Hsf and oxidative stress (Miller and Mittler, 2006). For example, HsfA1b is involved in the regulation of expression of APX2, which encodes cytosolic ascorbate peroxidase to scavenge hydrogen peroxide in the cytosol (Panchuk et al., 2002), and HsfA4a is a candidate for a sensor molecule involved in H2O2 perception in plants (Davletova et al., 2005). It has been shown that HsfA2 plays an important role in oxidative stress responses (Li et al., 2005). These findings indicate that HsfA2 participates in multiple stress responses. In fact, microarray data available to date show that the expression of HsfA2 is also up-regulated by salt and osmotic stress (Genevestigator, http://www.genevestigator.ethz.ch/at/). To confirm the microarray data, the expression level of HsfA2 was tested during salt and osmotic stress by semi-quantitative RT-PCR (Fig. 4A). The expression of HsfA2 was induced at 6 h after the NaCl treatment and peaked at 12 h. Then, the expression level was maintained at a relatively high level until 24 h after the treatment. The expression of HsfA2 was gradually induced until 24 h by osmotic stress treatment.
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To investigate salt and osmotic stress tolerance in the El2
::HsfA2 plants, the relative root length of wild-type and El2::HsfA2#24 plants grown on media containing NaCl or mannitol was compared. Figure 4B illustrates how inhibition of root elongation by NaCl or mannitol was significantly alleviated in the El2::HsfA2#24 plants in comparison to wild-type plants. Similar results were obtained in El2::HsfA2#25 plants (data not shown). As shown in Fig. 4C, the El2::HsfA2#24 plants exhibited growth defects compared with wild-type plants growing on control media (Fig. 1). However, shoots of El2::HsfA2#24 plants growing on media containing NaCl or mannitol were larger than those of wild-type plants (Fig. 4C). These results demonstrated that El2::HsfA2 plants were also involved in salt and osmotic stress responses. These findings indicate that HsfA2 functions in improving multiple stress tolerance.
Enhanced callus growth in the El2::HsfA2 plants
In animals, Hsfs are known to be involved in development, cell differentiation, and proliferation (Pirkkala et al., 2001). By contrast, it has not yet been reported in any plant that Hsfs play a role in cell differentiation and proliferation. Che et al. (2002) reported that expression of the HsfA2 gene increases during the process of callus formation and growth from root explants. Figure 5A shows that the mRNA level of the HsfA2 gene increased at 1 d on CIM, peaked at 3 d on CIM, and remained at a higher level than the control throughout the experimental period.
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An examination was carried out to see if the HsfA2 gene is involved in callus formation and growth in Arabidopsis. Figure 5B reveals that enhanced callus formation was apparently observed in root explants of the El2
::HsfA2#25 line at 7 d on CIM compared with wild types. Then, callus growth of the El2::HsfA2#25 line was significantly accelerated at 21 d on CIM (Fig. 5C). Figure 5C also shows that callus growth of the El2::HsfA2#19 line was weakly enhanced at 21 d on CIM compared with wild-type callus. Microarray analysis revealed that many stress-related genes were up-regulated during callus growth from root explants (Che et al., 2002). By contrast, callus growth of the El2::HsfA2C264 line was delayed (Fig. 5C). These results suggest that Hsfs play a role in cell proliferation. Since no obvious difference in callus growth was observed between wild type and the hsfA2 mutant (Fig. 5C), other class A Hsfs may be involved in callus formation and growth from root explants. In animals, Hsf is known to be required in not only the stress response but also development, cell differentiation, and proliferation (Pirkkala et al., 2001). Therefore, artificial elevation of expression of the HsfA2 genes could lead to enhanced stress tolerance, and at the same time to increased plant biomass in in vitro culture. Microarray analysis also showed that other stress-responsive transcription factor genes were also up-regulated during the process of callus growth from root explants (Che et al., 2006). It may be that some of these genes are also involved in not only the stress response but also cell differentiation and proliferation.
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Acknowledgements |
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We thank Drs Y Ohashi and I Mitsuhara (National Institute of Agrobiological Resources, Tsukuba, Japan) for providing the plasmid, pBE2113.
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Abbreviations |
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CIM, callus-inducing medium; CTAD, C-terminal activation domain; DBD, DNA binding domain; HR-A/B, hydrophobic coiled-coil region; HS, heat shock; HSE, heat shock element; Hsf, heat shock transcription factor; Hsp, heat shock protein.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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