Expression of rice heat stress transcription factor OsHsfA2e enhances ...

Planta (2008) 227:957–967 DOI 10.1007/s00425-007-0670-4

O R I G I N A L A R T I CL E

Expression of rice heat stress transcription factor OsHsfA2e enhances tolerance to environmental stresses in transgenic Arabidopsis Naoki Yokotani · Takanari Ichikawa · Youichi Kondou · Minami Matsui · Hirohiko Hirochika · Masaki Iwabuchi · Kenji Oda

Received: 25 September 2007 / Accepted: 12 November 2007 / Published online: 7 December 2007 © Springer-Verlag 2007

Abstract Plant growth and crop yields are limited by high-temperature stresses. In this study, we attempted to isolate the rice genes responsible for high-temperature stress tolerance using a transformed Arabidopsis population expressing a full-length cDNA library of rice. From approximately 20,000 lines of transgenic Arabidopsis, we isolated a thermotolerant line, R04333, that could survive transient heat stress at the cotyledon stage. The rice cDNA inserted in R04333 encodes OsHsfA2e, a member of the heat stress transcription factors. The thermotolerant phenotype was observed in newly constructed transgenic Arabidopsis plants expressing OsHsfA2e. Among 5 A2-type HSF genes encoded in the rice genome, four genes, including OsHsfA2e, are induced by high temperatures in rice seed-

lings. The OsHsfA2e protein was localized to the nuclear region and exhibited transcription activation activity in the C-terminal region. Microarray analysis demonstrated that under unstressed conditions transgenic Arabidopsis overexpressing OsHsfA2e highly expressed certain stress-associated genes, including several classes of heat-shock proteins. The thermotolerant phenotype was observed not only in the cotyledons but also in rosette leaves, inXorescence stems and seeds. In addition, transgenic Arabidopsis exhibited tolerance to high-salinity stress. These observations suggest that the OsHsfA2e may be useful in molecular breeding designed to improve the environmental stress tolerance of crops. Keywords Arabidopsis · FOX-hunting system · Heat stress transcription factor · High-temperature stress · Rice

Electronic supplementary material The online version of this article (doi:10.1007/s00425-007-0670-4) contains supplementary material, which is available to authorized users. N. Yokotani · M. Iwabuchi · K. Oda (&) Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan e-mail: [email protected] T. Ichikawa · Y. Kondou · M. Matsui Plant Functional Genomics Research Team, Plant Functional Genomic Research Group, Plant Science Center, RIKEN Yokohama Institute, 1-7-22 Suehirocho, Tsurumiku, Yokohama, Kanagawa 230-0045, Japan H. Hirochika Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki 305-8602, Japan

Abbreviations DBD DNA-binding domain FOX Full-length cDNA overexpressing GFP Green Xuorescent protein HSF Heat stress transcription factor HSP Heat-shock protein OE Overexpression

Introduction High temperature is one of the major abiotic stresses limiting plant yield and distribution worldwide. The major events of response to high-temperature stress are perception and transduction of stress signals through signalling components, resulting in the activation of stress-related genes and the synthesis of diverse functional proteins. A number

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of functional genes that respond to high-temperature stress have been described (Sung et al. 2003). For example, various types of heat-shock proteins (HSPs), synthesized under conditions of high temperature, have been proposed to act as molecular chaperones. Knockout or silencing of a heatinduced Arabidopsis HSP101 caused loss of the acquired thermotolerance, whereas the overexpression of HSP101 in transgenic plants improved tolerance to high-temperature stress (Hong et al. 2000; Queitsch et al. 2000). Transgenic plants overexpressing several classes of heat-induced small HSPs improved tolerance to environmental stresses, including heat stress (Scharf et al. 2001; Sun et al. 2001; Sanmiya et al. 2004; Neta-Sharir et al. 2005). Furthermore, under high-temperature conditions, the expression of enzymes synthesizing osmoprotective substances and antioxidant systems is increased (Panchuk et al. 2002; Panikulangara et al. 2004; Rizhsky et al. 2004). The expression of functional genes is largely regulated by speciWc transcription factors. Heat stress transcription factors (HSFs) play a central role in the heat-shock response in many species. HSFs bind to the heat-shock element (HSE) present in the promoter of many heat-inducible genes (Barros et al. 1992). Drosophila, Caenorhabditis elegans and yeast have a single HSF gene, whereas vertebrates have four HSF genes in their genome. In contrast, higher plants possess a large family of HSFs (Nover et al. 1996; Nakai 1999; Baniwal et al. 2004)-21 HSF genes in Arabidopsis (Nover et al. 2001), at least 23 in rice (Oryza sativa) and 18 in tomato (Lycopersicon esculentum; Baniwal et al. 2004; Kotak et al. 2004). Transgenic Arabidopsis overexpressing HSF genes exhibited up-regulation of stressassociated genes and enhanced thermotolerance (Lee et al. 1995; Prändl et al. 1998; Li et al. 2005; Nishizawa et al. 2006). Recent studies have suggested that constitutively expressed AtHsfA1a and b are required for the early expression of stress-associated genes, whereas heat-inducible AtHsfA2 is the major regulator of the acquired thermotolerance in Arabidopsis (Lohmann et al. 2004; Charng et al. 2007). In tomato, LeHsfA1 is expressed constitutively, whereas LeHsfA2 responds to high temperatures. The overexpression of LeHsfA1 improved the tolerance to high temperature stresses, whereas the silencing of LeHsfA1 caused a reduction in the thermotolerance in transgenic tomato plants (Mishra et al. 2002); these tomato plants exhibited a reduced expression of LeHsfA2 under heat stress conditions. The expression of LeHsfA2 in tomato protoplasts with silenced LeHsfA1 restored the thermotolerant phenotype (Mishra et al. 2002). The ERF/AP2 transcription factor DREB2A involved in dehydration and salinity responses (Liu et al. 1998) is also induced by high temperatures in Arabidopsis. The constitutive activation of DREB2A resulted in an enhanced tolerance to high-temperature stresses, whereas the knockout mutation of DREB2A

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caused a reduction in the acquired thermotolerance (Sakuma et al. 2006). In addition, the overexpression of the bZIP-type transcription factor ABF3 involved in abscisic acid (ABA) signalling induced high-temperature tolerance in transgenic Arabidopsis (Kim et al. 2004). Rice is one of the most important food crops in the world, and its complete genome has recently been sequenced (International Rice Genome Sequencing Project 2005). However, compared to Arabidopsis, there have been few studies investigating the stress-response-associated genes in rice, partly because of the longer life cycle of rice and the larger plant size. Recently, a novel system called the full-length cDNA overexpressing gene hunting (FOX hunting) system was developed (Ichikawa et al. 2006). This system uses Arabidopsis to facilitate rapid and large-scale functional analyses of genes. We utilized this system in order to identify rice genes involved in thermotolerance. In this study, we report the isolation and characterization of the rice A2 type HSF gene OsHsfA2e. We also report that the overexpression of OsHsfA2e can confer marked thermotolerance on Arabidopsis in several aspects such as vegetative growth, Xower stem elongation, and seed stage.

Materials and methods Plant growth conditions and stress treatment Arabidopsis thaliana ecotype Col-0 (obtained from Lehle Seeds, Tuscon, AZ, USA) was used as the wild-type. Plants were grown on soil or 1/2 Murashige and Skoog (MS) medium containing 1% sucrose and 0.8% agar under continuous light at 22°C. For performing heat stress treatment on the plants grown on the agar medium, a petri dish containing 4-day-old Arabidopsis seedlings was sealed with vinyl tape and dipped into water warmed to 42°C for 90 min. For performing heat stress treatment on plants grown in soil, the Arabidopsis plants were grown in soil covered by a nylon mesh in a Xowerpot and these were dipped inverted into water warmed to 45°C for 10 min. In order to test the seed thermotolerance, dry seeds were immersed at 50, 52 and 54°C for 30 min. After imbibition, the seeds were surface sterilized before sowing. In order to observe the eVect of high salinity on germination and greening, seeds were sown on agar medium containing 100, 125 or 150 mM NaCl and grown at 22°C under continuous light. In order to determine the eVects of NaCl at the vegetative stage, 9-day-old plants grown on an agar medium lacking NaCl were transferred to an agar medium containing 160 or 170 mM NaCl. Three days after the transfer, the plants were returned to agar medium lacking NaCl and grown for 4 days.

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For real-time RT-PCR analysis, seeds of rice (O. sativa L.) cv. Nipponbare (kindly gifted from Dr. Mori, National Institute of Agrobiological Sciences, Tsukuba, Japan) were sown on an MS medium containing 1% sucrose and 0.02% gellan gum and grown for 5 days in capped test tubes. The tubes were sealed with vinyl tape and dipped into hot water maintained at 42°C. Plant transformation In order to construct retransformation lines expressing OsHsfA2e, full-length cDNA fragments of OsHsfA2e were cloned into the SWI site of pBIG2113SF (Ichikawa et al. 2006) and introduced into Agrobacterium GV3101. Arabidopsis transformation was performed by the Xoral dipping method (Clough and Bent 1998). Transformed T1 seedlings were selected on a medium containing 1 mM KNO3, 50 mg¡1 hygromycin and 0.8% agar (Nakazawa and Matsui 2003). Estimation of mRNA by real-time RT-PCR Total RNA was isolated by using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), including a DNA elimination step. Here, 2
g of the total RNA was used for the cDNA synthesis using the ProstarTM Wrst strand RT-PCR kit (Stratagene, La Jolla, CA, USA) with a random hexamer primer. RT-PCR was performed using 25
l reaction mixtures containing 2
l of a 20-fold dilution of the reverse-transcribed cDNA, 2.5 mM of each primer and 12.5
l of SYBR Green PCR Master Mix (Applied Biosystems, Scoresby, VIA, Australia) with the ABI PRISM 7700 sequence detection system (Applied Biosystems). The sequences of the primers used are listed in supplementary Table S1. As a standard, quantitated PCR fragments ampliWed with gene-speciWc primers were used. The thermal cycling conditions were as follows: 40 cycles of 15 s at 95°C and 1 min at 60°C after pre-incubation at 95°C for 10 min. The amounts of the transcripts of rice and Arabidopsis genes were normalized by dividing the quantities of the transcripts by those of the coampliWed actin genes RAC1 and AtACT2, respectively (McElroy et al. 1990; An et al. 1996). Transient expression of GFP-OsHsfA2e in onion epidermal cells In order to express the N-terminal green Xuorescent protein (GFP)-fused OsHsfA2e under the control of the cauliXower mosaic virus 35S promoter, a GATEWAY (Invitrogen, Carlsbad, CA, USA) destination vector was constructed by inserting a HindIII–SacI fragment containing the 35S promoter, synthetic mutant GFP (Niwa et al. 1999) and GATEWAY cassette of pGWB6 (Tsuyoshi Nakagawa, Shimane University, Japan) into pBI221 (Clontech, Palo Alto, CA,

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USA). The coding sequence fragment of OsHsfA2e, which was ampliWed with primers containing a CACC sequence in front of the ATG start codon, was cloned into pENTR/DTOPO (Invitrogen) and transferred into the destination vector by the LR Clonase reaction according to the manufacturer’s protocol (Invitrogen). The transient expression of the GFPfusion protein was performed by the particle bombardment method using the PDS-1000/He particle delivery system (BioRad, Hercules, CA, USA). Here, 2
g of GFP-OsHsfA2e or GFP control plasmids was bombarded into onion epidermal cells. After an overnight incubation in the dark, the onion epidermis was examined under an LSM510 inverted confocal microscope (Zeiss, Oberkochen, Germany). The nuclei were stained with 4⬘,6-diamidino-2-phenylindole (DAPI) and visualized using UV light illumination. Transactivation analysis in yeast For the measurement of the transactivation activity, the complete or deleted OsHsfA2e coding sequence fragment ampliWed by PCR was cloned between the NdeI and PstI sites of pGBKT7 (Clontech). The recombinant plasmids were transformed into the yeast strain AH109 harbouring the LacZ and HIS3 reporter genes. The HIS3 reporter gene activity was conWrmed by a viability test on a medium lacking histidine. The LacZ activity was observed in a medium containing 250 mg/l X-gal. Microarray analysis Total RNA was extracted by using the RNeasy Plant Mini Kit (Qiagen). Biotin-labelled complementary RNA was prepared from 15
g of the total RNA and hybridized to an ATH1 oligonucleotide array according to the manufacturer’s protocol (AVymetrix, Santa Clara, CA). GeneChips were scanned using a Scanner 3000 and the resulting expression data were analysed by the GeneChip operating system (AVymetrix). Two independent biological replicates were carried out. The normalization of the raw data and the estimation of signal intensities were performed using Robust Multichip Average methodology (Bolstad et al. 2003). The average expression values and their Holmadjusted P values were calculated using the aVylmGUI package running on the R program (Irizarry et al. 2003).

Results Screening of rice FOX lines for tolerance to high-temperature stress We constructed approximately 20,000 lines of Arabidopsis (rice FOX lines) transformed by the Xoral dip method with

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In order to identify the transgene(s) introduced into R04333, we determined the nucleotide sequence of DNA fragments ampliWed by genomic PCR with speciWc primers for the 35S promoter and the nopaline synthase terminator according to the protocol of Ichikawa et al. (2006). The characterized sequence corresponded to a full-length cDNA clone of rice-AK068660. A Southern-blot analysis of the hygromycin-resistance gene HPTII from the genomic DNA extracted from a mixture of approximately 20 of the T2 plants revealed 2 bands, and all of the 25 independent T2 lines exhibited hygromycin resistance (data not

shown). In addition, all of the 95 independent T2 lines were positive transformants, as detected by genomic PCR with speciWc primers for AK068660 (data not shown). These results indicate that R04333 has two or more copies of a transgene. The cDNA of rice AK068660 encodes a putative heat stress transcription factor previously named OsHsfA2e (Baniwal et al. 2004; Kotak et al. 2004). In order to demonstrate the direct relationship between OsHsfA2e and the thermotolerant phenotype of R04333, we performed retransformation experiments (Ichikawa et al. 2006). Here, 12 out of the 14 reconstructed Arabidopsis plants overexpressing OsHsfA2e (retransformed lines) exhibited thermotolerance in the T2 generation similar to R04333 (data not shown). Because the original R04333 had a multicopy transgene, homozygous T3 generations of the retransformants with a single-copy transgene (OsHsfA2eOE#02 or OsHsfA2eOE#04) were isolated and used for the following experiments. Figure 1d shows the thermotolerant phenotypes of OsHsfA2eOE#02 and OsHsfA2eOE#04. OsHsfA2eOE#02 and OsHsfA2eOE#04 expressed OsHsfA2e mRNA at almost the same level as R04333, whereas the wild-type did not (Fig. 1c). OsHsfA2eOE#02 and OsHsfA2eOE#04 exhibited slightly retarded growth with dark green leaves similar to R04333 (Fig. 1e). These observations indicate that the phenotype of R04333 was caused by the overexpression of OsHsfA2e.

Fig. 1 Isolation and characterization of rice FOX lines exhibiting tolerance to high-temperature stresses. a Screening for high-temperature stress-tolerant lines. Approximately 15 seeds of each rice FOX line were grown on agar medium at 22°C for 4 days and then incubated at 45°C for 90 min. The photograph was taken 7 days after heat treatment. The surviving spot indicates the thermotolerant line R04333. b Alignment of the deduced amino acid sequences of OsHsfA2e and other HsfA2s from rice and Arabidopsis by CLUSTALW. Asterisks mark identical amino acids. The important domains of OsHsfA2e are indicated as follows: DBD, DNA-binding domain (yellow); NLS nuclear localization signals (purple); HR-A and HR-B hydrophobic heptad repeat region for oligomerization A and B (green); AHA aromatic and

large hydrophobic amino acid residues embedded in an acidic context (blue). c Estimation of OsHsfA2e transcripts in wild-type, original R04333 line and two retransformed lines (OsHsfA2eOE#02 and OsHsfA2eOE#04). Each mRNA was prepared from 15 seedlings grown on agar medium for 4 days. The original R04333 line and retransformed lines were used at the T2 and T3 generations, respectively. Relative mRNA levels were expressed as a percentage of the R04333 mRNA levels. d Thermotolerant phenotype of the original R04333 and retransformed lines. Four-day-old seedlings were incubated at 45°C for 90 min. The photograph was taken 7 days after treatment. e Twelveday-old wild-type, OsHsfA2eOE#02 and OsHsfA2eOE#04 seedlings grown on agar medium. Bar = 1 cm

an Agrobacterium library containing approximately 13,000 types of rice full-length cDNAs. The rice FOX library constructed was thus not saturated. In order to identify the rice genes involved in thermotolerance, we used transient heat stress to screen 4-day-old T2 seedlings of rice FOX lines grown on agar medium. Three candidate lines exhibiting high survivability were selected from the approximate 20,000 FOX lines. In particular, R04333 exhibited the most dramatic phenotype for high-temperature stress tolerance (Fig. 1a). This line exhibited no obvious morphological change without slightly slow growth and dark green leaves (data not shown). The thermotolerant phenotype of R04333 is caused by overexpression of the rice HSF gene OsHsfA2e

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The deduced amino acid sequence revealed that OsHsfA2e belongs to the class-A HSF subgroup. OsHsfA2e has an N-terminal DNA-binding domain (DBD) followed by an oligomerization domain with a hydrophobic heptad repeat region (HR-A and B) as in the other known class-A HSFs (Fig. 1b). In the C-terminal region, there are two short peptide motifs enriched with aromatic and large hydrophobic amino acid residues embedded in an acidic surrounding (AHA) motif. OsHsfA2e has two nuclear localization signals (NLSs) at the C-terminus of the DBD and midway between HR-B and AHA. GFP-fused OsHsfA2e is localized in the nucleus The subcellular localization of OsHsfA2e was examined by expressing the fusion protein with GFP in onion epidermal cells. DNA constructs encoding the GFP-fused, full-length OsHsfA2e (GFP-OsHsfA2e) and GFP under the control of the 35S promoter were introduced into the onion epidermal cells by particle bombardment. The GFP-OsHsfA2e signal was detected mainly in the nucleus, while the control GFP was detected in both the nucleus and the cytosol (Fig. 2a). This result indicates that the OsHsfA2e protein is targetted to the nucleus. Arabidopsis AtHsfA2 has a nuclear export signal (NES) at the extreme C-terminus, and the mutation of this motif causes an enhanced localization in the nucleus (Kotak et al. 2004). However, the GFP-fused OsHsfA2e without C-terminus was located at nucleus as well as full-length OsHsfA2e (data not shown) suggesting that OsHsfA2e has no NES in C-terminus unlike Arabidopsis AtHsfA2.

Fig. 2 Nuclear localization and transactivation activity of the OsHsfA2e protein. a Nuclear localization of OsHsfA2e in onion epidermal cells. GFP or N-terminal GFP-fused OsHsfA2e (GFP-OsHsfA2e) were transiently expressed in onion epidermal cells and observed using a confocal microscope. Left panels show the Xuorescent images of GFP and right panels show those of DAPI-stained nuclei. Arrow indicates

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OsHsfA2e has transactivation activity In order to test whether OsHsfA2e has a transactivation domain, we used a yeast assay system. The full-length or partial coding sequences of OsHsfA2e was fused to the GAL4 DBD and introduced into the yeast reporter strain AH109. The HIS3 reporter gene activity was conWrmed by a viability test on medium lacking histidine. In the absence of histidine, the yeasts transformed by both the construct containing the full-length OsHsfA2e and the constructs containing the amino acid region 210–357 or 305–357 of OsHsfA2e were able to survive (Fig. 2b). The LacZ activity of each yeast transformant was consistent with its HIS3 activity (Fig. 2b). These results indicate that OsHsfA2e exhibits transactivation activity in the region of amino acids 305– 357. Rice A2-type HSFs including OsHsfA2e expressed under high-temperature stress Recent studies have suggested that among the large family of HSF genes in higher plants, the A1- and A2-type HSFs play important roles in the high-temperature response in Arabidopsis. In the rice genome, 1 A1-type gene (OsHsfA1) and 5 A2-type genes (OsHsfA2a to e) have been identiWed. Among these, only OsHsfA2c (OsHsf6) has been reported to exhibit high-temperature-dependent induction (Liu et al. 2005). We analysed the expression patterns of all of the rice HsfA1 and HsfA2 genes under high-temperature stress conditions using real-time RT-PCR (Fig. 3). The expressions of OsHsfA2c, d and e increased within 1 h in response to

the corresponding nuclei with the GFP signal. b Transactivation assay of OsHsfA2e in yeast cells. The full-length construct and several partial deletion constructs of OsHsfA2e were fused to GAL4 DBD and expressed in the yeast strain AH109. The transformed yeasts were grown in a medium with or without histidine. The LacZ activity was observed in a medium containing X-gal

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Fig. 3 Expression of rice A1and A2-type HSFs under hightemperature stress conditions. Five-day-old rice seedlings were incubated at 42°C. Transcripts of each gene were measured using real-time RT-PCR. The relative quantity of each OsHsf mRNA was expressed as a percentage of OsHsfA2e at 42°C for 1 h. Data are expressed as the mean values § SD of three replicates

high temperature, and then decreased, whereas the expression of OsHsfA2b exhibited a gradual increase. In contrast, the transcripts of OsHsfA1 and OsHsfA2a did not increase under the high-temperature conditions. These results implied that OsHsfA2e is responsible for the heat stress response similar to that of OsHsfA2b, c and d. Since the responsiveness of OsHsfA2c was extremely high among all of the A2-type HSFs, it is likely that OsHsfA2c plays a major role in the heat stress response in rice. Overexpression of OsHsfA2e caused constitutive expression of stress-related genes In order to determine the OsHsfA2e target genes, we compared the gene expression proWles among 4-day-old seedlings of wild-type Col-0, OsHsfA2eOE#02 and OsHsfA2eOE#04 using the AVymetrix GeneChip ATH1. In the OsHsfA2eOE#02 plants, the expressions of 37 genes were fourfold higher than those of the wild-type and the expression of one gene was a quarter less (Holm-adjusted P value < 0.005). Table 1 lists the up-regulated genes in the OsHsfA2eOE#02 plants. The OsHsfA2eOE#04 plants exhibited almost identical results (Supplementary Table S2). Among these up-regulated genes, 22 genes were also induced by high-temperature treatment (Tables 1, supplementary Table S3). Many of the up-regulated genes are stress-associated genes including several types of HSPs. The expression levels of seven stress-associated genesHSP17.7-CII, HSP26.5-P, HSP70, HSP101, HSP17.6A-CI, HSP17.4-CIII and GolS1-were measured by real-time PCR (data not shown). Although all these genes were heatresponsive, only HSP17.7-CII, HSP26.5-P, HSP70 and GolS1 were up-regulated in the OsHsfA2eOE plants. Transgenic Arabidopsis overexpressing OsHsfA2e exhibited thermotolerance in leaves, inXorescence stems and seeds In order to estimate the agricultural usefulness of OsHsfA2e for molecular breeding, we tested the thermotolerance of

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vegetative stage, soil-grown plants. After transient heat shock at 45°C, most of the wild-type plants died, whereas all of the OsHsfA2eOE#02 and OsHsfA2eOE#04 plants survived (Fig. 4a). Thermotolerance was also observed in the inXorescence stem. After heat treatment at 45°C for 10 min, wild-type inXorescences wilted the next day and could not be restored within at least 2 weeks (Fig. 4b). On the other hand, although the inXorescences of OsHsfA2eOE plants stopped growing after the heat treatment, growth was restored 3 days after the heat treatment and plants developed normal Xowers after 1 week (Fig. 4c). Stem elongation 6 days after heat treatment was 5.6 § 4.5 mm for Col0, and 52.6 § 29.4 mm for OsHsfA2e#2 (mean value § SD, n = 7). In addition, we tested the thermotolerance of the transgenic Arabidopsis seed expressing OsHsfA2e. High temperatures during imbibition dramatically suppressed the germination of wild-type seeds, whereas transgenic seeds exhibited thermotolerance (Fig. 5). Overexpression of OsHsfA2e causes salt tolerance in transgenic Arabidopsis Among the up-regulated genes in OsHsfA2eOE plants (Table 1) are several genes involved not only in the response to high-temperature stress but also to various other types of environmental stress. This suggested that OsHsfA2eOE plants may be tolerant to environmental stresses other than heat stress. Thus, we compared salt tolerance in wild-type and OsHsfA2eOE plants by growing them on an agar medium containing NaCl. The timing of germination-the point at which the tip of the radical had fully penetrated the seed coat-was comparable (data not shown). However, the suppression of chlorophyll synthesis and cotyledon opening under salinity stress was less marked in the OsHsfA2eOE plants than in the wild-type. As shown in Fig. 6a, on medium containing 100 mM NaCl, nearly half of the wild-type seedlings exhibited chlorosis and stunted growth, whereas approximately 80% of the OsHsfA2eOE#02 and OsHsfA2eOE#04 plants developed green cotyledons. On medium containing 125 mM NaCl,

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Table 1 Up-regulated genes in OsHsfA2eOE plants identiWed by GeneChip analysis AGI No.

Gene function

Fold change

a

At4g27670

Hsp25.3-P

200.01

+

At3g46230

Hsp17.4-CI

87.60

+

At5g12030

Hsp17.7-CII

80.80

+

At4g10250

Hsp22.0-ER

51.45

+

At5g59720

Hsp18.1-CI

51.38

+

At1g53540

Hsp17.6C-CI

38.96

+

At1g52560

Hsp26.5-P

38.57

+

At1g25210

Unknown/UDP-3-0-acyl N-acetylglucosamine deacetylase family protein

26.71

At2g47180

Galactinol synthase (GolS1)

26.22

At3g47250

Expressed protein

24.33

At5g12020

HSP17.6-CII

22.91

At3g28270

Expressed protein

22.72

Heat inducible

+ +

At1g23960

Expressed protein

17.20

At3g44630

Disease resistance protein RPP1-WsB-like (TIR-NBS-LRR class)

16.84 13.05

+

11.61

+

At3g09640

L-ascorbate

At2g29500

Hsp17.6B-CI

peroxidase (APX2)

At3g28290

Integrin-related protein (14A)

11.24

At1g73490

RRM-containing protein

10.75

At5g41700

Ubiquitin-conjugating enzyme 8 (UBC8)

10.36

At5g59310

Lipid transfer protein 4 (LTP4)

9.56

At3g50970

Dehydrin/low-temperature-induced protein (XERO2/LTI30)

9.29

At2g32120

Hsp70T-2

9.10

+

At4g23493

Expressed protein

8.92

+

At5g48570

Peptidyl-prolyl cis–trans isomerase, putative

8.69

+

At3g47290

Phosphoinositide-speciWc phospholipase C family protein

8.28

At3g43740

Leucine-rich repeat family protein

8.18

At5g47610

Zinc Wnger (C3HC4-type RING Wnger) family protein

7.85

+

At4g28390

ADP, ATP carrier protein (AAC3)

6.90

+

At5g65390

Arabinogalactan-protein (AGP7)

6.25

At1g16030

Hsp70B

6.11

+ +

At3g12580

Hsp70

5.86

At4g02540

DC1 domain-containing protein

5.79

At4g21320

(2R)-Phospho-3-sulfolactate synthase-related

5.27

At1g03070

Low similarity to N-methyl-D-aspartate receptor-associated protein

4.25

+

At1g75750

Gibberellin-regulated protein 1 (GASA1)

4.22

+

At2g05440

Similar to glycine-rich protein

4.18

At4g12400

Stress-inducible protein, putative, similar to sti

4.14

+

+

The genes in the OsHsfA2eOE#02 plants with a fourfold higher expression than in wild-type are shown (Holm-adjusted P value < 0.005) a The genes increased fourfold by high-temperature treatment (42°C for 1 h)

all of the wild-type seedlings exhibited stunted growth; however, approximately 20% of the seedlings of the OsHsfA2eOE lines survived. Furthermore, we tested the eVects of NaCl at the vegetative stage. Nine-day-old plants grown on agar medium lacking NaCl were transferred onto agar medium containing 160 or 170 mM NaCl. After 3 days, almost all or all of the wild-type plants had died, whereas more than half of the OsHsfA2eOE#02 and OsHsfA2eOE#04 survived (Fig. 6b),

suggesting that the overexpression of OsHsfA2e improves the tolerance of transgenic plants to high salinity stress.

Discussion To date, several thermotolerance-related genes have been isolated using genetic approaches. For example, Hong and Vierling (2000) isolated seven mutants (hot1–hot7) defec-

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tive in the acquisition of thermotolerance and revealed that hot1 is HSP101. Further, atts02-another mutant lacking acquired thermotolerance-encodes digalactosyldiacylglycerol synthase 1 (DGD1; Burke et al. 2000; Chen et al. 2006). In addition, HIT1, characterized by heat-sensitive mutants encodes the homologue of yeast Vsp53p involved in vesicle traYcking (Lee et al. 2006). However, these mutants were characterized by defects in thermotolerance, and there are very few reports on screening stress-tolerant mutants. This implies that environmental stress tolerance is mainly associated with a gain-of-function phenotype and is therefore diYcult to characterize using knockout mutagenesis. In this study, we utilized the FOX hunting system

(Ichikawa et al. 2006) to isolate the rice genes involved in the tolerance to high-temperature stress. The OsHsfA2e gene isolated in the present study belongs to the class-A family of HSFs. The OsHsfA2e protein is localized in the nucleus (Fig. 2a). Further, OsHsfA2e functions as the transcriptional activator in yeast (Fig. 2b). A deletion analysis revealed that the activation domain of OsHsfA2e is localized in the 305–357 region (Fig. 2b)-a region that contains an AHA motif (Kotak et al. 2004). Most of the class-A HSFs have one or more AHA motifs that possess transactivation potential, whereas class-B and class-C HSFs do not (Kotak et al. 2004). Although tomato LeHsfA5 belongs to the class-A HSFs, it acts as a transrepressor that represses the activator activity of LeHsfA4b (Baniwal et al. 2007). Our observations suggest that the AHA motif of OsHsfA2e acts as a transactivation domain similar to that in most of the class-A HSFs. Although more than 21 HSF genes have been found in the rice genome, little is known about their individual roles. In view of the high genetic redundancy of HSFs in plants, it will probably be diYcult to reveal their individual molecular regulatory mechanisms. In Arabidopsis, the expression of a single A2-type HSF gene, AtHsfA2, increases substantially under high-temperature conditions (Busch et al. 2005; Li et al. 2005; Nishizawa et al. 2006). Similarly, in tomato, the expression of LeHsfA2 is induced by high temperatures (Mishra et al. 2002). In both Arabidopsis and tomatoes, the up-regulation of a single A2-type HSF gene improved the tolerance to high-temperature stress, and its down-regulation generated the opposite phenotype (Li et al. 2005; Charng et al. 2007; Mishra et al. 2002). In particular, many studies on Arabidopsis have demonstrated the importance of AtHsfA2 on the environmental stress response. The expression of AtHsfA2 is strongly induced by high temperatures (Busch et al. 2005; Li et al. 2005; Nishizawa et al.

Fig. 5 Thermotolerance of seeds. a Germination rate of wild-type and OsHsfA2eOE seeds after heat shock. Dry seeds immersed in hot water were sown on agar medium. After incubation at 4°C for 2 days, the seeds were incubated at 22°C for 6 days and germination rates were

measured. Each experiment was performed in triplicate with 21 seeds. Data are the mean values § SD for three individual experiments. b The state of germination of heat-treated seeds. Photograph was taken 6 days after heat shock at 54°C for 30 min

Fig. 4 Thermotolerance of soil-grown plants. a Thermotolerance at the vegetative stage. Fourteen-day-old plants were dipped into hot water maintained at 45°C for 10 min. Plants 4 days after heat shock are shown. Values indicate the survival rate (n = 40). b, c Thermotolerance of the inXorescence stem. InXorescence stems of 5- to 6-week-oldplants were dipped into hot water maintained at 45°C for 10 min. Representative wild-type (b) and OsHsfA2e#02 (c) inXorescence stems 4 days after heat shock are shown. Arrows indicate the approximate position where the top of the inXorescence was located at the time of heat treatment. Each experiment was replicated at least four times

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Fig. 6 Tolerance to high salinity stress. a EVects of high salinity on greening. Seeds were sown on agar medium containing NaCl and grown for 10 days at 22°C under continuous light. Each experiment was performed in triplicate with 15 seeds. Data are the mean values § SD for three individual experiments. b EVects of high salinity at the vegetative stage. Nine-day-old plants grown on an agar medium lacking NaCl were transferred to agar containing 160 or 170 mM NaCl. At 3 days after the transfer, the plants were returned to agar lacking NaCl. Plants 4 days after the salt treatment are shown. The experiment was repeated at least three times and similar results were obtained

2006). The overexpression of AtHsfA2 in transgenic Arabidopsis increases tolerance to the combined environmental stresses whereas the knockout mutant of AtHsfA2 causes a reduced expression of some heat-responsive genes and acquired thermotolerance (Li et al. 2005; Schramm et al. 2006; Nishizawa et al. 2006; Charng et al. 2007). On the basis of these results, AtHsfA2 has been suggested to be the major HSF gene responsible for the high-temperature response in Arabidopsis. We revealed that OsHsfA2e responds to high temperatures and induces enhanced stress tolerance in transgenic Arabidopsis similar to AtHsfA2. This suggests that rice OsHsfA2e might be a functional homologue of AtHsfA2. However, rice has at least Wve A2type HSF genes (OsHsfA2a to e; Baniwal et al. 2004; Kotak et al. 2004). Our expression data revealed that OsHsfA2b, c and d also increase within 1 h following exposure to high temperature (Fig. 3). In particular, the expression level of OsHsfA2c was more than 20- to 30-fold times higher than those of other OsHsfAs. Liu et al. (2005) also reported that the expression of OsHsfA2c (OsHsf6) is a response to high temperatures. As a result of screening, we isolated two fur-

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ther candidate FOX lines as thermotolerant lines. We found that another thermotolerant line, R13150, possessed OsHsfA2b and Arabidopsis retransformed with OsHsfA2b exhibited thermotolerance (data not shown). These observations suggest that 4 A2-type HSFs (OsHsfA2b, c, d and e) play a role in the response to high-temperature stress and that there is accordingly a degree of redundancy in this response. Moreover, Yamanouchi et al. (2002) reported that the mutation in another rice HSF gene, OsHsfA4d, causes a lesion mimic phenotype of rice spotted leaf (spl7) mutant plants. Interestingly, the spl7 mutation not only aVects pathogen susceptibility but also causes hypersensitivity to mild environmental stresses such as high temperatures and high light at the adult stage (Yamanouchi et al. 2002). The present study suggests that OsHsfA4d/SPL7 is a candidate for one of the most important HSFs with regard to the environmental stress response. Further study will be required to elucidate the individual roles of rice HSFs. In order to reveal the role of rice HSFs, particularly in rice, transgenic rice plants expressing OsHsfs will yield more information. Our microarray analysis revealed that 37 genes were upregulated in Arabidopsis overexpressing OsHsfA2e. Among these, 22 were heat-inducible (Table 1). The genes were mainly stress-associated genes including several types of HSPs. Since overexpression of HSPs, such as HSP70 or small HSPs, induces stress tolerance in transgenic plants (Sun et al. 2001; Sung and Guy 2003; Sanmiya et al. 2004; Neta-Sharir et al. 2005), it is likely that the thermotolerant phenotype of Arabidopsis overexpressing OsHsfA2e is caused by the constitutive expression of these stress-associated genes. Nishizawa et al. (2006) demonstrated that 46 genes were constitutively up-regulated in transgenic Arabidopsis overexpressing AtHsfA2. In both OsHsfA2e- and AtHsfA2-overexpressing plants, stress-associated genes such as HSPs, probably involving thermotolerance, were highly expressed. However, 31 out of 46 AtHsfA2-targetted genes are not increased in OsHsfA2e-overexpressing Arabidopsis. Most of these genes are functionally uncharacterized. The diVerence of target genes between OsHsfA2e and AtHsfA2 may be caused simply by the diVerence of experimental conditions such as plant stage or growth conditions. Otherwise, this may imply that the target sequence of A2-type HSFs is slightly diVerent between rice and Arabidopsis. It is also possible that these AtHsfA2-regulated genes are induced not by OsHsfA2e but by other members of the rice A2-type HSFs. Soil-grown Arabidopsis expressing OsHsfA2e exhibited improved tolerance to transient heat stresses at the vegetative and Xowering stages (Fig. 4). However, no signiWcant tolerance to continuous and moderate heat stresses (35°C) at the vegetative or Xowering stages was observed (data not shown). Tolerance to this treatment may be caused by

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acquired thermotolerance because Arabidopsis can develop acquired thermotolerance after exposure to 35–38°C for 1 h (Burke et al. 2000; Hong and Vierling 2000; Queitsch et al. 2000). Similarly, in transgenic Arabidopsis overexpressing AtHsfA1a (AtHsf1) or AtHsfAb (AtHsf3) an improved basal thermotolerance was found, but not acquired thermotolerance (Lee et al. 1995; Prändl et al. 1998). In the present study, plants overexpressing OsHsfA2e also exhibited thermotolerance at the seed stage (Fig. 5). Similarly, seed thermotolerance had been observed in transgenic tobacco expressing sunXower HaHsfA9 (Prieto-Dapena et al. 2006). It has been reported that the several types of HSPs increase during seed maturation (Wehmeyer et al. 1996; Wehmeyer and Vierling 2000). Recently, Kotak et al. (2007) found that the accumulation of HSPs during the late stage of seed development is regulated by AtHsfA9 in Arabidopsis. Whether OsHsfA2e is involved in thermotolerance in rice seeds should be elucidated by further studies. Overexpression of OsHsfA2e in Arabidopsis also resulted in enhanced tolerance to high salinity (Fig. 6). Several genes encoding proteins probably responsible for the adaptation to high salinity or osmotic stress are up-regulated by the overexpression of OsHsfA2e. For example, the molecular chaperone Hsp17.7-CII (Hsp17.6A) responds to heat, NaCl and high osmotic pressures and induces enhanced tolerance to salt and osmotic stresses upon overexpression in transgenic Arabidopsis (Sun et al. 2001). GolS1 is also induced by several environmental stresses and its overexpression in Arabidopsis induces water stress tolerance by virtue of high raYnose accumulation (Taji et al. 2002). It is likely that the salt-tolerant phenotype of Arabidopsis expressing OsHsfA2e is caused by the constitutive expression of these genes. Arabidopsis overexpressing the OsHsfA2e gene isolated by the FOX hunting system exhibited improved thermotolerance at various growth stages and in various tissues. In addition, the overexpression of OsHsfA2e conferred tolerance to high salinity. These observations suggest that the OsHsfA2e gene may be useful in molecular breeding designed to increase the tolerance to various types of environmental stress. Acknowledgments We thank Dr. Francis M. Mathooko (Jomo Kenyatta University, Kenya) for the careful perusal of this manuscript. We also thank Dr T. Nakagawa (Shimane University, Japan) for providing the pGWB6 plasmid and Dr M. Mori (National Institute of Agrobiological Sciences) for providing the rice seed. This work was supported in part by Special Coordination Funds for Promoting Science and Technology (Science and Technology Agency of Japan).

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